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1
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Zhou Q, Catalán P, Bell H, Baumann P, Cooke R, Evans R, Yang J, Zhang Z, Zappalà D, Zhang Y, Blackburn GM, He Y, Jin Y. An Ion-Pair Induced Intermediate Complex Captured in Class D Carbapenemase Reveals Chloride Ion as a Janus Effector Modulating Activity. ACS CENTRAL SCIENCE 2023; 9:2339-2349. [PMID: 38161376 PMCID: PMC10755735 DOI: 10.1021/acscentsci.3c00609] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/17/2023] [Revised: 11/17/2023] [Accepted: 11/17/2023] [Indexed: 01/03/2024]
Abstract
Antibiotic-resistant Enterobacterales that produce oxacillinase (OXA)-48-like Class D β-lactamases are often linked to increased clinical mortality. Though the catalytic mechanism of OXA-48 is known, the molecular origin of its biphasic kinetics has been elusive. We here identify selective chloride binding rather than decarbamylation of the carbamylated lysine as the source of biphasic kinetics, utilizing isothermal titration calorimetry (ITC) to monitor the complete reaction course with the OXA-48 variant having a chemically stable N-acetyl lysine. Further structural investigation enables us to capture an unprecedented inactive acyl intermediate wedged in place by a halide ion paired with a conserved active site arginine. Supported by mutagenesis and mathematical simulation, we identify chloride as a "Janus effector" that operates by allosteric activation of the burst phase and by inhibition of the steady state in kinetic assays of β-lactams. We show that chloride-induced biphasic kinetics directly affects antibiotic efficacy and facilitates the differentiation of clinical isolates encoding Class D from Class A and B carbapenemases. As chloride is present in laboratory and clinical procedures, our discovery greatly expands the roles of chloride in modulating enzyme catalysis and highlights its potential impact on the pharmacokinetics and efficacy of antibiotics during in vivo treatment.
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Affiliation(s)
- Qi Zhou
- Key
Laboratory of Synthetic and Natural Functional Molecule, College of
Chemistry and Materials Science, Northwest
University, Xi’an 710127, P. R. China
| | - Pablo Catalán
- Grupo
Interdisciplinar de Sistemas Complejos, Departamento de Matemáticas, Universidad Carlos III de Madrid, 28911 Leganés, Spain
| | - Helen Bell
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
| | - Patrick Baumann
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
- Manchester
Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom
| | - Rebekah Cooke
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
| | - Rhodri Evans
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
- Manchester
Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom
| | - Jianhua Yang
- Key
Laboratory of Synthetic and Natural Functional Molecule, College of
Chemistry and Materials Science, Northwest
University, Xi’an 710127, P. R. China
| | - Zhen Zhang
- Key
Laboratory of Synthetic and Natural Functional Molecule, College of
Chemistry and Materials Science, Northwest
University, Xi’an 710127, P. R. China
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
| | - Davide Zappalà
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
| | - Ye Zhang
- Key
Laboratory of Synthetic and Natural Functional Molecule, College of
Chemistry and Materials Science, Northwest
University, Xi’an 710127, P. R. China
| | - George Michael Blackburn
- School
of Biosciences, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom
| | - Yuan He
- Key
Laboratory of Synthetic and Natural Functional Molecule, College of
Chemistry and Materials Science, Northwest
University, Xi’an 710127, P. R. China
| | - Yi Jin
- School
of Chemistry, Cardiff University, Cardiff, CF10 3AT, United Kingdom
- Manchester
Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester M1 7DN, United Kingdom
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2
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Jain D, Verma J, Ajith T, Bhattacharjee A, Ghosh AS. Two non-active site residues W165 and L166 prominently influence the beta-lactam hydrolytic ability of OXA-23 beta-lactamase. J Antibiot (Tokyo) 2023; 76:489-498. [PMID: 37095236 DOI: 10.1038/s41429-023-00624-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2023] [Revised: 03/28/2023] [Accepted: 04/02/2023] [Indexed: 04/26/2023]
Abstract
Dissemination of class D OXA-type carbapenemases is one of the significant causes of beta-lactam resistance in Gram-negative bacteria. The amino acid residues present near the active site are involved in hydrolytic mechanism of class D carbapenemases, though it is not identified in OXA-23. Here, with the help of site-directed mutagenesis, we aimed to explicate the importance of the residues W165, L166 and V167 of the possible omega loop and residue D222 in the short β5-β6 loop on the activity of OXA-23. All the residues were substituted with alanine. The resultant proteins were assayed for the changes in activity in E. coli cells and purified for in vitro activity, and stability assessment. E. coli cells harboring OXA-23_W165A and OXA-23_L166A, individually, exhibited a significant decrease in resistance towards beta-lactam antibiotics as compared to OXA-23. Further, purified OXA-23_W165A and OXA-23_L166A imparted about >4-fold decrease in catalytic efficiency and displayed reduced thermal stability as compared to OXA-23. Bocillin-FL binding assay revealed that W165A substitution results in improper N-carboxylation of K82, leading to deacylation deficient OXA-23. Therefore, we infer that the residue W165 maintains the integrity of N-carboxylated lysine (K82) of OXA-23 and the residue L166 might be responsible for properly orientating the antibiotic molecules.
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Affiliation(s)
- Diamond Jain
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India
| | - Jyoti Verma
- Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India
| | - Tejavath Ajith
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India
| | | | - Anindya Sundar Ghosh
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, 721302, West Bengal, India.
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3
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Avci FG, Tastekil I, Jaisi A, Ozbek Sarica P, Sariyar Akbulut B. A review on the mechanistic details of OXA enzymes of ESKAPE pathogens. Pathog Glob Health 2022; 117:219-234. [PMID: 35758005 PMCID: PMC10081068 DOI: 10.1080/20477724.2022.2088496] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/17/2022] Open
Abstract
The production of β-lactamases is a prevalent mechanism that poses serious pressure on the control of bacterial resistance. Furthermore, the unavoidable and alarming increase in the transmission of bacteria producing extended-spectrum β-lactamases complicates treatment alternatives with existing drugs and/or approaches. Class D β-lactamases, designated as OXA enzymes, are characterized by their activity specifically towards oxacillins. They are widely distributed among the ESKAPE bugs that are associated with antibiotic resistance and life-threatening hospital infections. The inadequacy of current β-lactamase inhibitors for conventional treatments of 'OXA' mediated infections confirms the necessity of new approaches. Here, the focus is on the mechanistic details of OXA-10, OXA-23, and OXA-48, commonly found in highly virulent and antibiotic-resistant pathogens Acinetobacter baumannii, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter spp. to describe their similarities and differences. Furthermore, this review contains a specific emphasis on structural and computational perspectives, which will be valuable to guide efforts in the design/discovery of a common single-molecule drug against ESKAPE pathogens.
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Affiliation(s)
- Fatma Gizem Avci
- Bioengineering Department, Uskudar University, Uskudar, 34662, Turkey
| | - Ilgaz Tastekil
- Bioengineering Department, Marmara University, Kadikoy, 34722, Turkey
| | - Amit Jaisi
- Drug and Cosmetics Excellence Center, School of Pharmacy, Walailak University, 80160, Nakhon Si Thammarat, Thailand
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4
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Jain D, Verma J, Ghosh AS. Deciphering the role of residues in the loops nearing the active site of OXA-58 in imparting beta-lactamase activity. MICROBIOLOGY (READING, ENGLAND) 2022; 168. [PMID: 35766983 DOI: 10.1099/mic.0.001203] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
The existence of OXA-58 carbapenemase alone or in combination with other beta-lactam resistance factors poses significant beta-lactam resistance. The exact mechanism of action of OXA type beta-lactamases is debatable due to the involvement of multiple residues within or outside the active site. In the present work, we have elucidated the relative role of residues present in the putative omega (W169, L170, K171) and β6-β7 (A226 and D228) loops on the activity of OXA-58 by substituting into alanine (and aspartate for A226) through site-directed mutagenesis. E. coli cells harbouring OXA-58, substituted at the putative omega loop, manifest a significant decrease in the beta-lactam resistance profile than that of the cells expressing OXA-58. Further, a reduction in the catalytic efficiency is observed for the purified variants of OXA-58 carrying individual substitutions in the putative omega loop than that of OXA-58. However, the addition of NaHCO3 (for carbamylation of K86) increases catalytic efficiency of the individual protein as revealed by nitrocefin hydrolysis assay and steady state kinetics. Moreover, W169A and K171A substitutions show significant effects on the thermal stability of OXA-58. Therefore, we conclude that the putative omega loop residues W169, L170 and K171, individually, have significant role in the activity and stability of OXA-58, mostly by stabilising carbamylated lysine of active site.
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Affiliation(s)
- Diamond Jain
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
| | - Jyoti Verma
- Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
| | - Anindya S Ghosh
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
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Verma J, Jain D, Mallik D, Ghosh AS. Comparative insight into the roles of the non active-site residues E169 and N173 in imparting the beta-lactamase activity of CTX-M-15. FEMS Microbiol Lett 2022; 369:6530193. [PMID: 35175332 DOI: 10.1093/femsle/fnac018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/29/2021] [Revised: 01/03/2022] [Accepted: 02/15/2022] [Indexed: 11/13/2022] Open
Abstract
CTX-M-15 is a major extended-spectrum beta-lactamase disseminated throughout the globe. The roles of amino acids present in the active-site are widely studied though little is known about the role of the amino acids lying at the close proximity of the CTX-M-15 active-site. Here, by using site-directed mutagenesis we attempted to decipher the role of individual amino acids lying outside the active-site in imparting the beta-lactamase activity of CTX-M-15. Based on the earlier evidence, three amino acid residues namely, Glu169, Asp173 and Arg277 were substituted with alanine. The antibiotic susceptibility of E. coli cells harboring E169A and N173A substituted CTX-M-15 were enhanced by ∼ >32 fold for penicillins and ∼ 4-32 fold for cephalosporins, in comparison to CTX-M-15. However, cells carrying CTX-M-15_R277A did not show a significant difference in antibiotic susceptibility as compared to the wild-type. Further, the catalytic efficiency of the purified CTX-M-15_E169A and CTX-M-15_N173A were compromised when compared with the efficient beta-lactam hydrolysis of purified CTX-M-15. Moreover, the thermal stability of the mutated proteins CTX-M-15_E169A and CTX-M-15_N173A were reduced as compared to the wild type CTX-M-15. Therefore, we conclude that E169 and N173 are crucial non-active-site amino acids that are able to govern the CTX-M-15 activity.
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Affiliation(s)
- Jyoti Verma
- Advanced Technology Development Centre, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
| | - Diamond Jain
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
| | - Dhriti Mallik
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
| | - Anindya S Ghosh
- Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur-721302, West Bengal, India
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6
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Chiou J, Cheng Q, Shum PTF, Wong MHY, Chan EWC, Chen S. Structural and Functional Characterization of OXA-48: Insight into Mechanism and Structural Basis of Substrate Recognition and Specificity. Int J Mol Sci 2021; 22:ijms222111480. [PMID: 34768916 PMCID: PMC8583920 DOI: 10.3390/ijms222111480] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 10/10/2021] [Accepted: 10/21/2021] [Indexed: 02/04/2023] Open
Abstract
Class D β-lactamase OXA-48 is widely distributed among Gram-negative bacteria and is an important determinant of resistance to the last-resort carbapenems. Nevertheless, the detailed mechanism by which this β-lactamase hydrolyzes its substrates remains poorly understood. In this study, the complex structures of OXA-48 and various β-lactams were modeled and the potential active site residues that may interact with various β-lactams were identified and characterized to elucidate their roles in OXA-48 substrate recognition. Four residues, namely S70, K73, S118, and K208 were found to be essential for OXA-48 to undergo catalytic hydrolysis of various penicillins and carbapenems both in vivo and in vitro. T209 was found to be important for hydrolysis of imipenem, whereas R250 played a major role in hydrolyzing ampicillin, imipenem, and meropenem most likely by forming a H-bond or salt-bridge between the side chain of these two residues and the carboxylate oxygen ions of the substrates. Analysis of the effect of substitution of alanine in two residues, W105 and L158, revealed their roles in mediating the activity of OXA-48. Our data show that these residues most likely undergo hydrophobic interaction with the R groups and the core structure of the β-lactam ring in penicillins and the carbapenems, respectively. Unlike OXA-58, mass spectrometry suggested a loss of the C6-hydroxyethyl group during hydrolysis of meropenem by OXA-48, which has never been demonstrated in Class D carbapenemases. Findings in this study provide comprehensive knowledge of the mechanism of the substrate recognition and catalysis of OXA-type β-lactamases.
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Affiliation(s)
- Jiachi Chiou
- State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China; (J.C.); (Q.C.); (P.T.-f.S.); (M.H.-y.W.); (E.W.-c.C.)
| | - Qipeng Cheng
- State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China; (J.C.); (Q.C.); (P.T.-f.S.); (M.H.-y.W.); (E.W.-c.C.)
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong, China
| | - Perry Tim-fat Shum
- State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China; (J.C.); (Q.C.); (P.T.-f.S.); (M.H.-y.W.); (E.W.-c.C.)
| | - Marcus Ho-yin Wong
- State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China; (J.C.); (Q.C.); (P.T.-f.S.); (M.H.-y.W.); (E.W.-c.C.)
| | - Edward Wai-chi Chan
- State Key Laboratory of Chiroscience, Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China; (J.C.); (Q.C.); (P.T.-f.S.); (M.H.-y.W.); (E.W.-c.C.)
| | - Sheng Chen
- Department of Infectious Diseases and Public Health, Jockey Club College of Veterinary Medicine and Life Sciences, City University of Hong Kong, Kowloon, Hong Kong, China
- Correspondence:
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Mora-Ochomogo M, Lohans CT. β-Lactam antibiotic targets and resistance mechanisms: from covalent inhibitors to substrates. RSC Med Chem 2021; 12:1623-1639. [PMID: 34778765 PMCID: PMC8528271 DOI: 10.1039/d1md00200g] [Citation(s) in RCA: 56] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/15/2021] [Accepted: 07/25/2021] [Indexed: 12/24/2022] Open
Abstract
The β-lactams are the most widely used antibacterial agents worldwide. These antibiotics, a group that includes the penicillins and cephalosporins, are covalent inhibitors that target bacterial penicillin-binding proteins and disrupt peptidoglycan synthesis. Bacteria can achieve resistance to β-lactams in several ways, including the production of serine β-lactamase enzymes. While β-lactams also covalently interact with serine β-lactamases, these enzymes are capable of deacylating this complex, treating the antibiotic as a substrate. In this tutorial-style review, we provide an overview of the β-lactam antibiotics, focusing on their covalent interactions with their target proteins and resistance mechanisms. We begin by describing the structurally diverse range of β-lactam antibiotics and β-lactamase inhibitors that are currently used as therapeutics. Then, we introduce the penicillin-binding proteins, describing their functions and structures, and highlighting their interactions with β-lactam antibiotics. We next describe the classes of serine β-lactamases, exploring some of the mechanisms by which they achieve the ability to degrade β-lactams. Finally, we introduce the l,d-transpeptidases, a group of bacterial enzymes involved in peptidoglycan synthesis which are also targeted by β-lactam antibiotics. Although resistance mechanisms are now prevalent for all antibiotics in this class, past successes in antibiotic development have at least delayed this onset of resistance. The β-lactams continue to be an essential tool for the treatment of infectious disease, and recent advances (e.g., β-lactamase inhibitor development) will continue to support their future use.
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Affiliation(s)
| | - Christopher T Lohans
- Department of Biomedical and Molecular Sciences, Queen's University Kingston ON K7L 3N6 Canada
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8
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Abstract
Class D β-lactamases are composed of 14 families and the majority of the member enzymes are included in the OXA family. The genes for class D β-lactamases are frequently identified in the chromosome as an intrinsic resistance determinant in environmental bacteria and a few of these are found in mobile genetic elements carried by clinically significant pathogens. The most dominant OXA family among class D β-lactamases is superheterogeneous and the family needs to have an updated scheme for grouping OXA subfamilies through phylogenetic analysis. The OXA enzymes, even the members within a subfamily, have a diverse spectrum of resistance. Such varied activity could be derived from their active sites, which are distinct from those of the other serine β-lactamases. Their substrate profile is determined according to the size and position of the P-, Ω- and β5-β6 loops, assembling the active-site channel, which is very hydrophobic. Also, amino acid substitutions occurring in critical structures may alter the range of hydrolysed substrates and one subfamily could include members belonging to several functional groups. This review aims to describe the current class D β-lactamases including the functional groups, occurrence types (intrinsic or acquired) and substrate spectra and, focusing on the major OXA family, a new model for subfamily grouping will be presented.
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Affiliation(s)
- Eun-Jeong Yoon
- Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, South Korea
| | - Seok Hoon Jeong
- Department of Laboratory Medicine and Research Institute of Bacterial Resistance, Yonsei University College of Medicine, Seoul, South Korea
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GC-MS Discrimination of Citrulline from Ornithine and Homocitrulline from Lysine by Chemical Derivatization: Evidence of Formation of N5-Carboxy-ornithine and N6-Carboxy-lysine. Molecules 2021; 26:molecules26082301. [PMID: 33921162 PMCID: PMC8071523 DOI: 10.3390/molecules26082301] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Revised: 04/09/2021] [Accepted: 04/13/2021] [Indexed: 12/01/2022] Open
Abstract
Derivatization of amino acids by 2 M HCl/CH3OH (60 min, 80 °C) followed by derivatization of the intermediate methyl esters with pentafluoropropionic anhydride (PFPA) in ethyl acetate (30 min, 65 °C) is a useful two-step derivatization procedure (procedure A) for their quantitative measurement in biological samples by gas chromatography-mass spectrometry (GC-MS) as methyl ester pentafluoropropionic (PFP) derivatives, (Me)m-(PFP)n. This procedure allows in situ preparation of trideutero-methyl esters PFP derivatives, (d3Me)m-(PFP)n, from synthetic amino acids and 2 M HCl/CD3OD for use as internal standards. However, procedure A converts citrulline (Cit) to ornithine (Orn) and homocitrulline (hCit) to lysine (Lys) due to the instability of their carbamide groups under the acidic conditions of the esterification step. In the present study, we investigated whether reversing the order of the two-step derivatization may allow discrimination and simultaneous analysis of these amino acids. Pentafluoropropionylation (30 min, 65 °C) and subsequent methyl esterification (30 min, 80 °C), i.e., procedure B, of Cit resulted in the formation of six open and cyclic reaction products. The most abundant product is likely to be N5-Carboxy-Orn. The second most abundant product was confirmed to be Orn. The most abundant reaction product of hCit was confirmed to be Lys, with the minor reaction product likely being N6-Carboxy-Lys. Mechanisms are proposed for the formation of the reaction products of Cit and hCit via procedure B. It is assumed that at the first derivatization step, amino acids form (N,O)-PFP derivatives including mixed anhydrides. At the second derivatization step, the Cit-(PFP)4 and hCit-(PFP)4 are esterified on their C1-Carboxylic groups and on their activated Nureido groups. Procedure B also allows in situ preparation of (d3Me)m-(PFP)n from synthetic amino acids for use as internal standards. It is demonstrated that the derivatization procedure B enables discrimination between Cit and Orn, and between hCit and Lys. The utility of procedure B to measure simultaneously these amino acids in biological samples such as plasma and urine remains to be demonstrated. Further work is required to optimize the derivatization conditions of procedure B for biological amino acids.
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De Angelis G, Del Giacomo P, Posteraro B, Sanguinetti M, Tumbarello M. Molecular Mechanisms, Epidemiology, and Clinical Importance of β-Lactam Resistance in Enterobacteriaceae. Int J Mol Sci 2020; 21:ijms21145090. [PMID: 32708513 PMCID: PMC7404273 DOI: 10.3390/ijms21145090] [Citation(s) in RCA: 84] [Impact Index Per Article: 16.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/27/2020] [Revised: 07/13/2020] [Accepted: 07/17/2020] [Indexed: 12/16/2022] Open
Abstract
Despite being members of gut microbiota, Enterobacteriaceae are associated with many severe infections such as bloodstream infections. The β-lactam drugs have been the cornerstone of antibiotic therapy for such infections. However, the overuse of these antibiotics has contributed to select β-lactam-resistant Enterobacteriaceae isolates, so that β-lactam resistance is nowadays a major concern worldwide. The production of enzymes that inactivate β-lactams, mainly extended-spectrum β-lactamases and carbapenemases, can confer multidrug resistance patterns that seriously compromise therapeutic options. Further, β-lactam resistance may result in increases in the drug toxicity, mortality, and healthcare costs associated with Enterobacteriaceae infections. Here, we summarize the updated evidence about the molecular mechanisms and epidemiology of β-lactamase-mediated β-lactam resistance in Enterobacteriaceae, and their potential impact on clinical outcomes of β-lactam-resistant Enterobacteriaceae infections.
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Affiliation(s)
- Giulia De Angelis
- Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (G.D.A.); (B.P.); (M.S.)
- Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy;
| | - Paola Del Giacomo
- Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy;
| | - Brunella Posteraro
- Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (G.D.A.); (B.P.); (M.S.)
- Dipartimento di Scienze Gastroenterologiche, Endocrino-Metaboliche e Nefro-Urologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy
| | - Maurizio Sanguinetti
- Dipartimento di Scienze Biotecnologiche di Base, Cliniche Intensivologiche e Perioperatorie, Università Cattolica del Sacro Cuore, 00168 Rome, Italy; (G.D.A.); (B.P.); (M.S.)
- Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy;
| | - Mario Tumbarello
- Dipartimento di Scienze di Laboratorio e Infettivologiche, Fondazione Policlinico Universitario A. Gemelli IRCCS, 00168 Rome, Italy;
- Dipartimento di Sicurezza e Bioetica, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
- Correspondence:
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11
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Stewart NK, Smith CA, Toth M, Stasyuk A, Vakulenko SB. The crystal structures of CDD-1, the intrinsic class D β-lactamase from the pathogenic Gram-positive bacterium Clostridioides difficile, and its complex with cefotaxime. J Struct Biol 2019; 208:107391. [PMID: 31550535 PMCID: PMC6903424 DOI: 10.1016/j.jsb.2019.09.008] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2019] [Revised: 09/18/2019] [Accepted: 09/20/2019] [Indexed: 02/04/2023]
Abstract
Class D β-lactamases, enzymes that degrade β-lactam antibiotics and are widely spread in Gram-negative bacteria, were for a long time not known in Gram-positive organisms. Recently, these enzymes were identified in various non-pathogenic Bacillus species and subsequently in Clostridioides difficile, a major clinical pathogen associated with high morbidity and mortality rates. Comparison of the BPU-1 enzyme from Bacillus pumilus with the CDD-1 and CDD-2 enzymes from C. difficile demonstrated that the latter enzymes have broadened their substrate profile to efficiently hydrolyze the expanded-spectrum methoxyimino cephalosporins, cefotaxime and ceftriaxone. These two antibiotics are major contributors to the development of C. difficile infection, as they suppress sensitive bacterial microflora in the gut but fail to kill the pathogen which is highly resistant to these drugs. To gain insight into the structural features that contribute to the expansion of the substrate profile of CDD enzymes compared to BPU-1, we solved the crystal structures of CDD-1 and its complex with cefotaxime. Comparison of CDD-1 structures with those of class D enzymes from Gram-negative bacteria showed that in the cefotaxime-CDD-1 complex, the antibiotic is bound in a substantially different mode due to structural differences in the enzymes' active sites. We also found that CDD-1 has a uniquely long Ω-loop when compared to all other class D β-lactamases. This Ω-loop extension allows it to engage in hydrogen bonding with the acylated cefotaxime, thus providing additional stabilizing interactions with the substrate which could be responsible for the high catalytic activity of the enzyme for expanded-spectrum cephalosporins.
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Affiliation(s)
- Nichole K Stewart
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
| | - Clyde A Smith
- Department of Chemistry, Stanford University, Stanford, CA, USA; Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA, USA.
| | - Marta Toth
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA
| | - Anastasiya Stasyuk
- Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA, USA
| | - Sergei B Vakulenko
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN, USA.
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12
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Tooke CL, Hinchliffe P, Bragginton EC, Colenso CK, Hirvonen VHA, Takebayashi Y, Spencer J. β-Lactamases and β-Lactamase Inhibitors in the 21st Century. J Mol Biol 2019; 431:3472-3500. [PMID: 30959050 PMCID: PMC6723624 DOI: 10.1016/j.jmb.2019.04.002] [Citation(s) in RCA: 519] [Impact Index Per Article: 86.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2019] [Revised: 03/27/2019] [Accepted: 04/01/2019] [Indexed: 12/31/2022]
Abstract
The β-lactams retain a central place in the antibacterial armamentarium. In Gram-negative bacteria, β-lactamase enzymes that hydrolyze the amide bond of the four-membered β-lactam ring are the primary resistance mechanism, with multiple enzymes disseminating on mobile genetic elements across opportunistic pathogens such as Enterobacteriaceae (e.g., Escherichia coli) and non-fermenting organisms (e.g., Pseudomonas aeruginosa). β-Lactamases divide into four classes; the active-site serine β-lactamases (classes A, C and D) and the zinc-dependent or metallo-β-lactamases (MBLs; class B). Here we review recent advances in mechanistic understanding of each class, focusing upon how growing numbers of crystal structures, in particular for β-lactam complexes, and methods such as neutron diffraction and molecular simulations, have improved understanding of the biochemistry of β-lactam breakdown. A second focus is β-lactamase interactions with carbapenems, as carbapenem-resistant bacteria are of grave clinical concern and carbapenem-hydrolyzing enzymes such as KPC (class A) NDM (class B) and OXA-48 (class D) are proliferating worldwide. An overview is provided of the changing landscape of β-lactamase inhibitors, exemplified by the introduction to the clinic of combinations of β-lactams with diazabicyclooctanone and cyclic boronate serine β-lactamase inhibitors, and of progress and strategies toward clinically useful MBL inhibitors. Despite the long history of β-lactamase research, we contend that issues including continuing unresolved questions around mechanism; opportunities afforded by new technologies such as serial femtosecond crystallography; the need for new inhibitors, particularly for MBLs; the likely impact of new β-lactam:inhibitor combinations and the continuing clinical importance of β-lactams mean that this remains a rewarding research area.
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Affiliation(s)
- Catherine L Tooke
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - Philip Hinchliffe
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - Eilis C Bragginton
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - Charlotte K Colenso
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - Viivi H A Hirvonen
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - Yuiko Takebayashi
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom
| | - James Spencer
- School of Cellular and Molecular Medicine, University of Bristol Biomedical Sciences Building, University Walk, Bristol BS8 1TD, United Kingdom.
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13
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Fluorescent Antibiotics: New Research Tools to Fight Antibiotic Resistance. Trends Biotechnol 2018; 36:523-536. [PMID: 29478675 DOI: 10.1016/j.tibtech.2018.01.004] [Citation(s) in RCA: 57] [Impact Index Per Article: 8.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2017] [Revised: 01/10/2018] [Accepted: 01/11/2018] [Indexed: 01/02/2023]
Abstract
Better understanding how multidrug-resistant (MDR) bacteria can evade current and novel antibiotics requires a better understanding of the chemical biology of antibiotic action. This necessitates using new tools and techniques to advance our knowledge of bacterial responses to antibiotics, ideally in live cells in real time, to selectively investigate bacterial growth, division, metabolism, and resistance in response to antibiotic challenge. In this review, we discuss the preparation and biological evaluation of fluorescent antibiotics, focussing on how these reporters and assay methods can help elucidate resistance mechanisms. We also examine the potential utility of such probes for real-time in vivo diagnosis of infections.
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14
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Multiple substitutions lead to increased loop flexibility and expanded specificity in Acinetobacter baumannii carbapenemase OXA-239. Biochem J 2018; 475:273-288. [PMID: 29229762 DOI: 10.1042/bcj20170702] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2017] [Revised: 12/07/2017] [Accepted: 12/11/2017] [Indexed: 11/17/2022]
Abstract
OXA-239 is a class D carbapenemase isolated from an Acinetobacter baumannii strain found in Mexico. This enzyme is a variant of OXA-23 with three amino acid substitutions in or near the active site. These substitutions cause OXA-239 to hydrolyze late-generation cephalosporins and the monobactam aztreonam with greater efficiency than OXA-23. OXA-239 activity against the carbapenems doripenem and imipenem is reduced ∼3-fold and 20-fold, respectively. Further analysis demonstrated that two of the substitutions (P225S and D222N) are largely responsible for the observed alteration of kinetic parameters, while the third (S109L) may serve to stabilize the protein. Structures of OXA-239 with cefotaxime, doripenem and imipenem bound as acyl-intermediates were determined. These structures reveal that OXA-239 has increased flexibility in a loop that contains P225S and D222N. When carbapenems are bound, the conformation of this loop is essentially identical with that observed previously for OXA-23, with a narrow active site that makes extensive contacts to the ligand. When cefotaxime is bound, the loop can adopt a different conformation that widens the active site to allow binding of that bulky drug. This alternate conformation is made possible by P225S and further stabilized by D222N. Taken together, these results suggest that the three substitutions were selected to expand the substrate specificity profile of OXA-23 to cephalosporins and monobactams. The loss of activity against imipenem, however, suggests that there may be limits to the plasticity of class D enzymes with regard to evolving active sites that can effectively bind multiple classes of β-lactam drugs.
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15
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Lund BA, Thomassen AM, Carlsen TJO, Leiros HKS. Structure, activity and thermostability investigations of OXA-163, OXA-181 and OXA-245 using biochemical analysis, crystal structures and differential scanning calorimetry analysis. Acta Crystallogr F Struct Biol Commun 2017; 73:579-587. [PMID: 28994407 PMCID: PMC5633926 DOI: 10.1107/s2053230x17013838] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/21/2017] [Accepted: 09/25/2017] [Indexed: 01/20/2023] Open
Abstract
The first crystal structures of the class D β-lactamases OXA-181 and OXA-245 were determined to 2.05 and 2.20 Å resolution, respectively; in addition, the structure of a new crystal form of OXA-163 was resolved to 2.07 Å resolution. All of these enzymes are OXA-48-like and have been isolated from different clinical Klebsiella pneumoniae strains and also from other human pathogens such as Pseudomonas aeruginosa and Escherichia coli. Here, enzyme kinetics and thermostability studies are presented, and the new crystal structures are used to explain the observed variations. OXA-245 had the highest melting point (Tm = 55.8°C), as determined by differential scanning calorimetry, compared with OXA-163 (Tm = 49.4°C) and OXA-181 (Tm = 52.6°C). The differences could be explained by the loss of two salt bridges in OXA-163, and an overall decrease in the polarity of the surface of OXA-181 compared with OXA-245.
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Affiliation(s)
- Bjarte Aarmo Lund
- Department of Chemistry, UiT The Arctic University of Norway, 9037 Tromsø, Norway
| | - Ane Molden Thomassen
- Department of Chemistry, UiT The Arctic University of Norway, 9037 Tromsø, Norway
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16
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June CM, Muckenthaler TJ, Schroder EC, Klamer ZL, Wawrzak Z, Powers RA, Szarecka A, Leonard DA. The structure of a doripenem-bound OXA-51 class D β-lactamase variant with enhanced carbapenemase activity. Protein Sci 2016; 25:2152-2163. [PMID: 27636561 DOI: 10.1002/pro.3040] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2016] [Revised: 09/09/2016] [Accepted: 09/09/2016] [Indexed: 11/10/2022]
Abstract
OXA-51 is a class D β-lactamase that is thought to be the native carbapenemase of Acinetobacter baumannii. Many variants of OXA-51 containing active site substitutions have been identified from A. baumannii isolates, and some of these substitutions increase hydrolytic activity toward carbapenem antibiotics. We have determined the high-resolution structures of apo OXA-51 and OXA-51 with one such substitution (I129L) with the carbapenem doripenem trapped in the active site as an acyl-intermediate. The structure shows that acyl-doripenem adopts an orientation very similar to carbapenem ligands observed in the active site of OXA-24/40 (doripenem) and OXA-23 (meropenem). In the OXA-51 variant/doripenem complex, the indole ring of W222 is oriented away from the doripenem binding site, thereby eliminating a clash that is predicted to occur in wildtype OXA-51. Similarly, in the OXA-51 variant complex, L129 adopts a different rotamer compared to I129 in wildtype OXA-51. This alternative position moves its side chain away from the hydroxyethyl moiety of doripenem and relieves another potential clash between the enzyme and carbapenem substrates. Molecular dynamics simulations of OXA-51 and OXA-51 I129L demonstrate that compared to isoleucine, a leucine at this position greatly favors a rotamer that accommodates the ligand. These results provide a molecular justification for how this substitution generates enhanced binding affinity for carbapenems, and therefore helps explain the prevalence of this substitution in clinical OXA-51 variants.
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Affiliation(s)
- Cynthia M June
- Department of Chemistry, Grand Valley State University, Allendale, Michigan, 49401
| | | | - Emma C Schroder
- Department of Chemistry, Grand Valley State University, Allendale, Michigan, 49401
| | - Zachary L Klamer
- Department of Cell and Molecular Biology, Grand Valley State University, Allendale, Michigan, 49401
| | - Zdzislaw Wawrzak
- Life Sciences Collaborative Access Team, Synchrotron Research Center, Northwestern University, Argonne, Illinois, 60439
| | - Rachel A Powers
- Department of Chemistry, Grand Valley State University, Allendale, Michigan, 49401
| | - Agnieszka Szarecka
- Department of Cell and Molecular Biology, Grand Valley State University, Allendale, Michigan, 49401
| | - David A Leonard
- Department of Chemistry, Grand Valley State University, Allendale, Michigan, 49401
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17
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Sirota FL, Maurer-Stroh S, Eisenhaber B, Eisenhaber F. Single-residue posttranslational modification sites at the N-terminus, C-terminus or in-between: To be or not to be exposed for enzyme access. Proteomics 2016; 15:2525-46. [PMID: 26038108 PMCID: PMC4745020 DOI: 10.1002/pmic.201400633] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2014] [Revised: 04/17/2015] [Accepted: 05/29/2015] [Indexed: 11/30/2022]
Abstract
Many protein posttranslational modifications (PTMs) are the result of an enzymatic reaction. The modifying enzyme has to recognize the substrate protein's sequence motif containing the residue(s) to be modified; thus, the enzyme's catalytic cleft engulfs these residue(s) and the respective sequence environment. This residue accessibility condition principally limits the range where enzymatic PTMs can occur in the protein sequence. Non‐globular, flexible, intrinsically disordered segments or large loops/accessible long side chains should be preferred whereas residues buried in the core of structures should be void of what we call canonical, enzyme‐generated PTMs. We investigate whether PTM sites annotated in UniProtKB (with MOD_RES/LIPID keys) are situated within sequence ranges that can be mapped to known 3D structures. We find that N‐ or C‐termini harbor essentially exclusively canonical PTMs. We also find that the overwhelming majority of all other PTMs are also canonical though, later in the protein's life cycle, the PTM sites can become buried due to complex formation. Among the remaining cases, some can be explained (i) with autocatalysis, (ii) with modification before folding or after temporary unfolding, or (iii) as products of interaction with small, diffusible reactants. Others require further research how these PTMs are mechanistically generated in vivo.
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Affiliation(s)
- Fernanda L Sirota
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore
| | - Sebastian Maurer-Stroh
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore.,School of Biological Sciences (SBS), Nanyang Technological University (NTU), Singapore
| | - Birgit Eisenhaber
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore
| | - Frank Eisenhaber
- Bioinformatics Institute (BII), Agency for Science and Technology (A*STAR), Matrix, Singapore.,Department of Biological Sciences (DBS), National University of Singapore (NUS), Singapore.,School of Computer Engineering (SCE), Nanyang Technological University (NTU), Singapore
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18
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Shapiro AB. Investigation of β-lactam antibacterial drugs, β-lactamases, and penicillin-binding proteins with fluorescence polarization and anisotropy: a review. Methods Appl Fluoresc 2016; 4:024002. [DOI: 10.1088/2050-6120/4/2/024002] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
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19
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Meziane-Cherif D, Bonnet R, Haouz A, Courvalin P. Structural insights into the loss of penicillinase and the gain of ceftazidimase activities by OXA-145 β-lactamase in Pseudomonas aeruginosa. J Antimicrob Chemother 2015; 71:395-402. [PMID: 26568564 DOI: 10.1093/jac/dkv375] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2015] [Accepted: 10/13/2015] [Indexed: 11/14/2022] Open
Abstract
OBJECTIVES We previously described extended-spectrum oxacillinase OXA-145 from Pseudomonas aeruginosa, which differs from narrow-spectrum OXA-35 by loss of Leu-155. The deletion results in loss of benzylpenicillin hydrolysis and acquisition of activity against ceftazidime. We report the crystal structure of OXA-145 and provide the basis of its switch in substrate specificity. METHODS OXA-145 variants were generated by site-directed mutagenesis and purified to homogeneity. The crystal structure of OXA-145 was determined and molecular dynamics simulations were performed. Kinetic parameters were investigated in the absence and in the presence of sodium hydrogen carbonate (NaHCO3) for representative substrates. RESULTS The structure of OXA-145 was obtained at a resolution of 2.3 Å and its superposition with that of OXA-10 showed that Trp-154 was shifted by 1.8 Å away from the catalytic Lys-70, which was not N-carboxylated. Addition of NaHCO3 significantly increased the catalytic efficiency against penicillins, but not against ceftazidime. The active-site cavity of OXA-145 was larger than that of OXA-10, which may favour the accommodation of large molecules such as ceftazidime. Molecular dynamics simulations of OXA-145 in complex with ceftazidime revealed two highly coordinated water molecules on the α- or β-face of the acyl ester bond, between Ser-67 and ceftazidime, which could be involved in the catalytic process. CONCLUSIONS Deletion of Leu-155 resulted in inefficient positioning of Trp-154, leading to a non-carboxylated Lys-70 and thus to loss of hydrolysis of the penicillins. Ceftazidime hydrolysis could be attributed to enlargement of the active site and to a catalytic mechanism independent of the carboxylated Lys-70.
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Affiliation(s)
- D Meziane-Cherif
- Institut Pasteur, Unité des Agents Antibactériens, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
| | - R Bonnet
- Laboratoire de bactériologie mycologie et parasitologie, Pôle de biologie médicale et d'anatomie pathologique, CHU de Clermont Ferrand - Hôpital Gabriel Montpied, 58 rue Montalembert, 63003 Clermont-Ferrand cedex 1, France
| | - A Haouz
- Institut Pasteur, Plateforme de cristallographie, CNRS-UMR3528, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
| | - P Courvalin
- Institut Pasteur, Unité des Agents Antibactériens, 25 rue du Docteur Roux, 75724 Paris cedex 15, France
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20
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Structural basis for carbapenem-hydrolyzing mechanisms of carbapenemases conferring antibiotic resistance. Int J Mol Sci 2015; 16:9654-92. [PMID: 25938965 PMCID: PMC4463611 DOI: 10.3390/ijms16059654] [Citation(s) in RCA: 120] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Revised: 04/21/2015] [Accepted: 04/22/2015] [Indexed: 02/06/2023] Open
Abstract
Carbapenems (imipenem, meropenem, biapenem, ertapenem, and doripenem) are β-lactam antimicrobial agents. Because carbapenems have the broadest spectra among all β-lactams and are primarily used to treat infections by multi-resistant Gram-negative bacteria, the emergence and spread of carbapenemases became a major public health concern. Carbapenemases are the most versatile family of β-lactamases that are able to hydrolyze carbapenems and many other β-lactams. According to the dependency of divalent cations for enzyme activation, carbapenemases can be divided into metallo-carbapenemases (zinc-dependent class B) and non-metallo-carbapenemases (zinc-independent classes A, C, and D). Many studies have provided various carbapenemase structures. Here we present a comprehensive and systematic review of three-dimensional structures of carbapenemase-carbapenem complexes as well as those of carbapenemases. We update recent studies in understanding the enzymatic mechanism of each class of carbapenemase, and summarize structural insights about regions and residues that are important in acquiring the carbapenemase activity.
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21
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Sahuquillo-Arce JM, Hernández-Cabezas A, Yarad-Auad F, Ibáñez-Martínez E, Falomir-Salcedo P, Ruiz-Gaitán A. Carbapenemases: A worldwide threat to antimicrobial therapy. World J Pharmacol 2015; 4:75-95. [DOI: 10.5497/wjp.v4.i1.75] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/29/2014] [Revised: 11/07/2014] [Accepted: 12/01/2014] [Indexed: 02/07/2023] Open
Abstract
Carbapenems are potent β-lactams with activity against extended-spectrum cephalosporinases and β-lactamases. These antibiotics, derived from thienamycn, a carbapenem produced by the environmental bacterium Streptomyces cattleya, were initially used as last-resort treatments for severe Gram-negative bacterial infections presenting resistance to most β-lactams but have become an empirical option in countries with high prevalence of Extended Spectrum β-lactamase-producing bacterial infections. Imipenem, the first commercially available carbapenem, was approved for clinical use in 1985. Since then, a wide variety of carbapenem-resistant bacteria has appeared, primarily Enterobacteriaceae such as Escherichia coli or Klebsiella pneumoniae (K. pneumoniae), Pseudomonas aeruginosa and Acinetobacter baumannii, presenting different resistance mechanisms. The most relevant mechanism is the production of carbapenem-hydrolyzing β-lactamases, also known as carbapenemases. These enzymes also inactivate all known β-lactams, and some of these enzymes can be acquired through horizontal gene transfer. Moreover, plasmids, transposons and integrons harboring these genes typically carry other resistance determinants, rendering the recipient bacteria resistant to almost all currently used antimicrobials, as is the case for K. pneumoniae carbapenemase - or New Delhi metallo-β-lactamases-type enzymes. The recent advent of these enzymes in the health landscape presents a serious challenge. First, the emergence of carbapenemases limits the currently available treatment options; second, these enzymes pose a risk to patients, as some studies have demonstrated high mortality associated with carbapenemase-producing bacterial infections; and third, these circumstances require an extra cost to sanitary systems, which are particularly cumbersome in developing countries. Therefore, emphasis should be placed on the early detection of these enzymes, the prevention of the spread of carbapenemase-producing bacteria and the development of new drugs resistant to carbapenemase hydrolysis.
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22
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Mitchell JM, Clasman JR, June CM, Kaitany KCJ, LaFleur JR, Taracila MA, Klinger NV, Bonomo RA, Wymore T, Szarecka A, Powers RA, Leonard DA. Structural basis of activity against aztreonam and extended spectrum cephalosporins for two carbapenem-hydrolyzing class D β-lactamases from Acinetobacter baumannii. Biochemistry 2015; 54:1976-87. [PMID: 25710192 DOI: 10.1021/bi501547k] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
The carbapenem-hydrolyzing class D β-lactamases OXA-23 and OXA-24/40 have emerged worldwide as causative agents for β-lactam antibiotic resistance in Acinetobacter species. Many variants of these enzymes have appeared clinically, including OXA-160 and OXA-225, which both contain a P → S substitution at homologous positions in the OXA-24/40 and OXA-23 backgrounds, respectively. We purified OXA-160 and OXA-225 and used steady-state kinetic analysis to compare the substrate profiles of these variants to their parental enzymes, OXA-24/40 and OXA-23. OXA-160 and OXA-225 possess greatly enhanced hydrolytic activities against aztreonam, ceftazidime, cefotaxime, and ceftriaxone when compared to OXA-24/40 and OXA-23. These enhanced activities are the result of much lower Km values, suggesting that the P → S substitution enhances the binding affinity of these drugs. We have determined the structures of the acylated forms of OXA-160 (with ceftazidime and aztreonam) and OXA-225 (ceftazidime). These structures show that the R1 oxyimino side-chain of these drugs occupies a space near the β5-β6 loop and the omega loop of the enzymes. The P → S substitution found in OXA-160 and OXA-225 results in a deviation of the β5-β6 loop, relieving the steric clash with the R1 side-chain carboxypropyl group of aztreonam and ceftazidime. These results reveal worrying trends in the enhancement of substrate spectrum of class D β-lactamases but may also provide a map for β-lactam improvement.
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Affiliation(s)
| | | | | | | | | | - Magdalena A Taracila
- ∥Departments of Medicine, Pharmacology, Biochemistry, and Molecular Biology and Microbiology, Case Western Reserve University and Research Service, and Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States
| | | | - Robert A Bonomo
- ∥Departments of Medicine, Pharmacology, Biochemistry, and Molecular Biology and Microbiology, Case Western Reserve University and Research Service, and Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio 44106, United States
| | - Troy Wymore
- ⊥UT/ORNL Center for Molecular Biophysics, Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, United States
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23
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Antunes NT, Fisher JF. Acquired Class D β-Lactamases. Antibiotics (Basel) 2014; 3:398-434. [PMID: 27025753 PMCID: PMC4790369 DOI: 10.3390/antibiotics3030398] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/03/2014] [Revised: 07/31/2014] [Accepted: 08/08/2014] [Indexed: 12/21/2022] Open
Abstract
The Class D β-lactamases have emerged as a prominent resistance mechanism against β-lactam antibiotics that previously had efficacy against infections caused by pathogenic bacteria, especially by Acinetobacter baumannii and the Enterobacteriaceae. The phenotypic and structural characteristics of these enzymes correlate to activities that are classified either as a narrow spectrum, an extended spectrum, or a carbapenemase spectrum. We focus on Class D β-lactamases that are carried on plasmids and, thus, present particular clinical concern. Following a historical perspective, the susceptibility and kinetics patterns of the important plasmid-encoded Class D β-lactamases and the mechanisms for mobilization of the chromosomal Class D β-lactamases are discussed.
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Affiliation(s)
- Nuno T Antunes
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
| | - Jed F Fisher
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA.
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24
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Che T, Bethel CR, Pusztai-Carey M, Bonomo RA, Carey PR. The different inhibition mechanisms of OXA-1 and OXA-24 β-lactamases are determined by the stability of active site carboxylated lysine. J Biol Chem 2014; 289:6152-64. [PMID: 24443569 DOI: 10.1074/jbc.m113.533562] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/10/2023] Open
Abstract
The catalytic efficiency of class D β-lactamases depends critically on an unusual carboxylated lysine as the general base residue for both the acylation and deacylation steps of the enzyme. Microbiological and biochemical studies on the class D β-lactamases OXA-1 and OXA-24 showed that the two enzymes behave differently when reacting with two 6-methylidene penems (penem 1 and penem 3): the penems are good inhibitors of OXA-1 but act more like substrates for OXA-24. UV difference and Raman spectroscopy revealed that the respective reaction mechanisms are different. The penems form an unusual intermediate, a 1,4-thiazepine derivative in OXA-1, and undergo deacylation followed by the decarboxylation of Lys-70, rendering OXA-1 inactive. This inactivation could not be reversed by the addition of 100 mM NaHCO3. In OXA-24, under mild conditions (enzyme:inhibitor = 1:4), only hydrolyzed products were detected, and the enzyme remained active. However, under harsh conditions (enzyme:inhibitor = 1:2000), OXA-24 was inhibited via decarboxylation of Lys-84; however, the enzyme could be reactivated by the addition of 100 mM NaHCO3. We conclude that OXA-24 not only decarboxylates with difficulty but also recarboxylates with ease; in contrast, OXA-1 decarboxylates easily but recarboxylates with difficulty. Structural analysis of the active site indicates that a crystallographic water molecule may play an important role in carboxylation in OXA-24 (an analogous water molecule is not found in OXA-1), supporting the suggestion that a water molecule in the active site of OXA-24 can lower the energy barrier for carboxylation significantly.
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Affiliation(s)
- Tao Che
- From the Departments of Biochemistry
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25
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Nikolaidis I, Favini-Stabile S, Dessen A. Resistance to antibiotics targeted to the bacterial cell wall. Protein Sci 2014; 23:243-59. [PMID: 24375653 DOI: 10.1002/pro.2414] [Citation(s) in RCA: 97] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/21/2013] [Revised: 12/21/2013] [Accepted: 12/23/2013] [Indexed: 11/10/2022]
Abstract
Peptidoglycan is the main component of the bacterial cell wall. It is a complex, three-dimensional mesh that surrounds the entire cell and is composed of strands of alternating glycan units crosslinked by short peptides. Its biosynthetic machinery has been, for the past five decades, a preferred target for the discovery of antibacterials. Synthesis of the peptidoglycan occurs sequentially within three cellular compartments (cytoplasm, membrane, and periplasm), and inhibitors of proteins that catalyze each stage have been identified, although not all are applicable for clinical use. A number of these antimicrobials, however, have been rendered inactive by resistance mechanisms. The employment of structural biology techniques has been instrumental in the understanding of such processes, as well as the development of strategies to overcome them. This review provides an overview of resistance mechanisms developed toward antibiotics that target bacterial cell wall precursors and its biosynthetic machinery. Strategies toward the development of novel inhibitors that could overcome resistance are also discussed.
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Affiliation(s)
- I Nikolaidis
- Institut de Biologie Structurale (IBS), Université Grenoble Alpes, 6 rue Jules Horowitz, 38027, Grenoble, France; Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Grenoble, France; Centre National de la Recherche Scientifique (CNRS), UMR 5075, Grenoble, France; Bijvoet Center for Biomolecular Research, Department of Biochemistry of Membranes, Utrecht University, Utrecht, The Netherlands
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Leonard DA, Bonomo RA, Powers RA. Class D β-lactamases: a reappraisal after five decades. Acc Chem Res 2013; 46:2407-15. [PMID: 23902256 DOI: 10.1021/ar300327a] [Citation(s) in RCA: 87] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
Despite 70 years of clinical use, β-lactam antibiotics still remain at the forefront of antimicrobial chemotherapy. The major challenge to these life-saving therapeutics is the presence of bacterial enzymes (i.e., β-lactamases) that can hydrolyze the β-lactam bond and inactivate the antibiotic. These enzymes can be grouped into four classes (A-D). Among the most genetically diverse are the class D β-lactamases. In this class are β-lactamases that can inactivate the entire spectrum of β-lactam antibiotics (penicillins, cephalosporins, and carbapenems). Class D β-lactamases are mostly found in Gram-negative bacteria such as Pseudomonas aeruginosa , Escherichia coli , Proteus mirabilis , and Acinetobacter baumannii . The active-sites of class D β-lactamases contain an unusual N-carboxylated lysine post-translational modification. A strongly hydrophobic active-site helps create the conditions that allow the lysine to combine with CO2, and the resulting carbamate is stabilized by a number of hydrogen bonds. The carboxy-lysine plays a symmetric role in the reaction, serving as a general base to activate the serine nucleophile in the acylation reaction, and the deacylating water in the second step. There are more than 250 class D β-lactamases described, and the full set of variants shows remarkable diversity with regard to substrate binding and turnover. Narrow-spectrum variants are most effective against the earliest generation penicillins and cephalosporins such as ampicillin and cephalothin. Extended-spectrum variants (also known as extended-spectrum β-lactamases, ESBLs) pose a more dangerous clinical threat as they possess a small number of substitutions that allow them to bind and hydrolyze later generation cephalosporins that contain bulkier side-chain constituents (e.g., cefotaxime, ceftazidime, and cefepime). Mutations that permit this versatility seem to cluster in the area surrounding an active-site tryptophan resulting in a widened active-site to accommodate the oxyimino side-chains of these cephalosporins. More concerning are the class D β-lactamases that hydrolyze clinically important carbapenem β-lactam drugs (e.g., imipenem). Whereas carbapenems irreversibly acylate and inhibit narrow-spectrum β-lactamases, class D carbapenemases are able to recruit and activate a deacylating water. The rotational orientation of the C6 hydroxyethyl group found on all carbapenem antibiotics likely plays a role in whether the deacylating water is effective or not. Inhibition of class D β-lactamases is a current challenge. Commercially available inhibitors that are active against other classes of β-lactamases are ineffective against class D enzymes. On the horizon are several compounds, consisting of both β-lactam derivatives and non-β-lactams, that have the potential of providing novel leads to design new mechanism-based inactivators that are effective against the class D enzymes. Several act synergistically when given in combination with a β-lactam antibiotic, and others show a unique mechanism of inhibition that is distinct from the traditional β-lactamase inhibitors. These studies will bolster structure-based inhibitor design efforts to facilitate the optimization and development of these compounds as class D inactivators.
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Affiliation(s)
- David A. Leonard
- Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401, United States
| | - Robert A. Bonomo
- Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, and Department of Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, United States
| | - Rachel A. Powers
- Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401, United States
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Structural origins of oxacillinase specificity in class D β-lactamases. Antimicrob Agents Chemother 2013; 58:333-41. [PMID: 24165180 DOI: 10.1128/aac.01483-13] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/25/2023] Open
Abstract
Since the discovery and use of penicillin, the increase of antibiotic resistance among bacterial pathogens has become a major health concern. The most prevalent resistance mechanism in Gram-negative bacteria is due to β-lactamase expression. Class D β-lactamases are of particular importance due to their presence in multidrug-resistant Acinetobacter baumannii and Pseudomonas aeruginosa. The class D enzymes were initially characterized by their ability to efficiently hydrolyze isoxazolyl-type β-lactams like oxacillin. Due to this substrate preference, these enzymes are traditionally referred to as oxacillinases or OXAs. However, this class is comprised of subfamilies characterized by diverse activities that include oxacillinase, carbapenemase, or cephalosporinase substrate specificity. OXA-1 represents one subtype of class D enzyme that efficiently hydrolyzes oxacillin, and OXA-24/40 represents another with weak oxacillinase, but increased carbapenemase, activity. To examine the structural basis for the substrate selectivity differences between OXA-1 and OXA-24/40, the X-ray crystal structures of deacylation-deficient mutants of these enzymes (Lys70Asp for OXA-1; Lys84Asp for OXA-24) in complexes with oxacillin were determined to 1.4 Å and 2.4 Å, respectively. In the OXA-24/40/oxacillin structure, the hydrophobic R1 side chain of oxacillin disrupts the bridge between Tyr112 and Met223 present in the apo OXA-24/40 structure, causing the main chain of the Met223-containing loop to adopt a completely different conformation. In contrast, in the OXA-1/oxacillin structure, a hydrophobic pocket consisting of Trp102, Met99, Phe217, Leu161, and Leu255 nicely complements oxacillin's nonpolar R1 side chain. Comparison of the OXA-1/oxacillin and OXA-24/40/oxacillin complexes provides novel insight on how substrate selectivity is achieved among subtypes of class D β-lactamases. By elucidating important active site interactions, these findings can also inform the design of novel antibiotics and inhibitors.
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Smith CA, Antunes NT, Stewart NK, Toth M, Kumarasiri M, Chang M, Mobashery S, Vakulenko SB. Structural basis for carbapenemase activity of the OXA-23 β-lactamase from Acinetobacter baumannii. ACTA ACUST UNITED AC 2013; 20:1107-15. [PMID: 24012371 DOI: 10.1016/j.chembiol.2013.07.015] [Citation(s) in RCA: 88] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/26/2013] [Revised: 07/23/2013] [Accepted: 07/28/2013] [Indexed: 11/18/2022]
Abstract
Dissemination of Acinetobacter baumannii strains harboring class D β-lactamases producing resistance to carbapenem antibiotics severely limits our ability to treat deadly Acinetobacter infections. Susceptibility determination in the A. baumannii background and kinetic studies with a homogeneous preparation of OXA-23 β-lactamase, the major carbapenemase present in A. baumannii, document the ability of this enzyme to manifest resistance to last-resort carbapenem antibiotics. We also report three X-ray structures of OXA-23: apo OXA-23 at two different pH values, and wild-type OXA-23 in complex with meropenem, a carbapenem substrate. The structures and dynamics simulations reveal an important role for Leu166, whose motion regulates the access of a hydrolytic water molecule to the acyl-enzyme species in imparting carbapenemase activity.
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Affiliation(s)
- Clyde A Smith
- Stanford Synchrotron Radiation Lightsource, Stanford University, Menlo Park, CA 94025, USA.
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29
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Shapiro AB, Gu RF, Gao N, Livchak S, Thresher J. Continuous fluorescence anisotropy-based assay of BOCILLIN FL penicillin reaction with penicillin binding protein 3. Anal Biochem 2013; 439:37-43. [PMID: 23603065 DOI: 10.1016/j.ab.2013.04.009] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2013] [Revised: 03/12/2013] [Accepted: 04/01/2013] [Indexed: 10/27/2022]
Abstract
We report a simple, rapid, and reproducible fluorescence anisotropy-based method for measuring rate constants for acylation and deacylation of soluble penicillin binding protein (PBP) constructs by compounds in microtiter plates by means of competition with time-dependent acylation by BOCILLIN FL. The method is demonstrated by measuring the acylation rate constants of the PBP3 periplasmic domains from Pseudomonas aeruginosa and Acinetobacter baumannii by BOCILLIN FL, aztreonam, meropenem, and ceftazidime. The new method requires very little protein and can be completed in approximately 1h per compound. A set of BOCILLIN FL acylation progress curves collected over a range of competitor concentrations is fit globally to a kinetic model by numerical integration. First-order deacylation rate constants could also be measured, as demonstrated with a catalytically impaired mutant OXA-10 β-lactamase.
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Affiliation(s)
- Adam B Shapiro
- Bioscience Department, Infection Innovative Medicines, AstraZeneca R&D Boston, Waltham, MA 02451, USA.
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30
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Abstract
Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many--and quite possibly all--bacteria, the peptidoglycan fragments are recovered and recycled. Although cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall-targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall-recycling inhibitors.
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Affiliation(s)
- Jarrod W Johnson
- Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA
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31
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Buchman JS, Schneider KD, Lloyd AR, Pavlish SL, Leonard DA. Site-saturation mutagenesis of position V117 in OXA-1 β-lactamase: effect of side chain polarity on enzyme carboxylation and substrate turnover. Biochemistry 2012; 51:3143-50. [PMID: 22429123 DOI: 10.1021/bi201896k] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Class D β-lactamases pose an emerging threat to the efficacy of β-lactam therapy for bacterial infections. Class D enzymes differ mechanistically from other β-lactamases by the presence of an active-site N-carboxylated lysine that serves as a general base to activate the serine nucleophile for attack. We have used site-saturation mutagenesis at position V117 in the class D β-lactamase OXA-1 to investigate how alterations in the environment around N-carboxylated K70 affect the ability of that modified residue to carry out its normal function. Minimum inhibitory concentration analysis of the 20 position 117 variants demonstrates a clear pattern of charge and polarity effects on the level of ampicillin resistance imparted on Escherichia coli (E. coli). Substitutions that introduce a negative charge (D, E) at position 117 reduce resistance to near background levels, while the positively charged K and R residues maintain the highest resistance levels of all mutants. Treatment of the acidic variants with the fluorescent penicillin BOCILLIN FL followed by SDS-PAGE shows that they are active for acylation by substrate but deacylation-deficient. We used a novel fluorescence anisotropy assay to show that the specific charge and hydrogen-bonding potential of the residue at position 117 affect CO(2) binding to K70, which in turn correlates to deacylation activity. These conclusions are discussed in light of the mechanisms proposed for both class D β-lactamases and BlaR β-lactam sensor proteins and suggest a reason for the preponderance of asparagine at the V117-homologous position in the sensors.
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Affiliation(s)
- Jennifer S Buchman
- Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401, USA
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32
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Bush K, Fisher JF. Epidemiological expansion, structural studies, and clinical challenges of new β-lactamases from gram-negative bacteria. Annu Rev Microbiol 2012; 65:455-78. [PMID: 21740228 DOI: 10.1146/annurev-micro-090110-102911] [Citation(s) in RCA: 306] [Impact Index Per Article: 23.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
β-Lactamase evolution presents to the infectious disease community a major challenge in the treatment of infections caused by multidrug-resistant gram-negative bacteria. Because over 1,000 of these naturally occurring β-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum β-lactamase (ESBL) families are frequently identified, the older CTX-M-14 and CTX-M-15 enzymes have become the most prevalent ESBLs in global surveillance. Carbapenemases with either serine-based or zinc-facilitated hydrolysis mechanisms are posing some of the most critical problems. Most geographical regions now report KPC serine carbapenemases and the metallo-β-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple β-lactamases appearing in a single organism, makes the design of new β-lactamase inactivators or β-lactamase-stable β-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.
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Affiliation(s)
- Karen Bush
- Biology Department, Indiana University, Bloomington, Indiana 47401, USA.
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Böttcher T, Sieber SA. β-Lactams and β-lactones as activity-based probes in chemical biology. MEDCHEMCOMM 2012. [DOI: 10.1039/c2md00275b] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
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Szarecka A, Lesnock KR, Ramirez-Mondragon CA, Nicholas HB, Wymore T. The Class D beta-lactamase family: residues governing the maintenance and diversity of function. Protein Eng Des Sel 2011; 24:801-9. [PMID: 21859796 PMCID: PMC3170078 DOI: 10.1093/protein/gzr041] [Citation(s) in RCA: 21] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/03/2011] [Revised: 07/14/2011] [Accepted: 07/26/2011] [Indexed: 12/29/2022] Open
Abstract
Class D β-lactamases, a major source of bacterial resistance to β-lactam antibiotic therapies, represent a distinct subset of the β-lactamase superfamily. They share a serine hydrolase mechanism with Classes A/C vs. Class B. Further understanding of their sequence-structure-function relationships would benefit efforts to design a new generation of antibiotics as well as to predict evolutionary mechanisms in response to such therapies. Here we describe analyses based on our high-resolution multiple sequence alignment and phylogenetic tree of ∼80 Class D β-lactamases that leverage several 3D structures of these enzymes. We observe several sequence clusters on the phylogenetic tree, some that are species specific while others include several species from α-, β- and γ-proteobacteria. Residues characteristic of a specific cluster were identified and shown to be located just outside the active site, possibly modulating the function of the catalytic residues to facilitate reactions with specific types of β-lactams. Most significant was the discovery of a likely disulfide bond in a large group composed of α-, β- and γ-proteobacteria that would contribute to enzyme stability and hence bacterial viability under antibiotic assault. A network of co-evolving residues was identified which suggested the importance of maintaining a surface for binding a highly conserved Phe69.
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Affiliation(s)
- Agnieszka Szarecka
- Department of Cell and Molecular Biology, Grand Valley State University, Henry Hall, 1 Campus Drive, Allendale, MI 49401, USA
| | - Kimberly R. Lesnock
- National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center, 300 South Craig Street, Pittsburgh, PA 15215, USA
| | - Carlos A. Ramirez-Mondragon
- National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center, 300 South Craig Street, Pittsburgh, PA 15215, USA
| | - Hugh B. Nicholas
- National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center, 300 South Craig Street, Pittsburgh, PA 15215, USA
| | - Troy Wymore
- National Resource for Biomedical Supercomputing, Pittsburgh Supercomputing Center, 300 South Craig Street, Pittsburgh, PA 15215, USA
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Verma V, Testero SA, Amini K, Wei W, Liu J, Balachandran N, Monoharan T, Stynes S, Kotra LP, Golemi-Kotra D. Hydrolytic mechanism of OXA-58 enzyme, a carbapenem-hydrolyzing class D β-lactamase from Acinetobacter baumannii. J Biol Chem 2011; 286:37292-303. [PMID: 21880707 DOI: 10.1074/jbc.m111.280115] [Citation(s) in RCA: 33] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Carbapenem-hydrolyzing class D β-lactamases (CHDLs) represent an emerging antibiotic resistance mechanism encountered among the most opportunistic Gram-negative bacterial pathogens. We report here the substrate kinetics and mechanistic characterization of a prominent CHDL, the OXA-58 enzyme, from Acinetobacter baumannii. OXA-58 uses a carbamylated lysine to activate the nucleophilic serine used for β-lactam hydrolysis. The deacylating water molecule approaches the acyl-enzyme species, anchored at this serine (Ser-83), from the α-face. Our data show that OXA-58 retains the catalytic machinery found in class D β-lactamases, of which OXA-10 is representative. Comparison of the homology model of OXA-58 and the recently solved crystal structures of OXA-24 and OXA-48 with the OXA-10 crystal structure suggests that these CHDLs have evolved the ability to hydrolyze imipenem, an important carbapenem in clinical use, by subtle structural changes in the active site. These changes may contribute to tighter binding of imipenem to the active site and removal of steric hindrances from the path of the deacylating water molecule.
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Affiliation(s)
- Vidhu Verma
- Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada
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36
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Structures of the class D carbapenemase OXA-24 from Acinetobacter baumannii in complex with doripenem. J Mol Biol 2011; 406:583-94. [PMID: 21215758 DOI: 10.1016/j.jmb.2010.12.042] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2010] [Revised: 12/24/2010] [Accepted: 12/29/2010] [Indexed: 11/20/2022]
Abstract
The emergence of class D β-lactamases with carbapenemase activity presents an enormous challenge to health practitioners, particularly with regard to the treatment of infections caused by Gram-negative pathogens such as Acinetobacter baumannii. Unfortunately, class D β-lactamases with carbapenemase activity are resistant to β-lactamase inhibitors. To better understand the details of the how these enzymes bind and hydrolyze carbapenems, we have determined the structures of two deacylation-deficient variants (K84D and V130D) of the class D carbapenemase OXA-24 with doripenem bound as a covalent acyl-enzyme intermediate. Doripenem adopts essentially the same configuration in both OXA-24 variant structures, but varies significantly when compared to the non-carbapenemase class D member OXA-1/doripenem complex. The alcohol of the 6α hydroxyethyl moiety is directed away from the general base carboxy-K84, with implications for activation of the deacylating water. The tunnel formed by the Y112/M223 bridge in the apo form of OXA-24 is largely unchanged by the binding of doripenem. The presence of this bridge, however, causes the distal pyrrolidine/sulfonamide group to bind in a drastically different conformation compared to doripenem bound to OXA-1. The resulting difference in the position of the side-chain bridge sulfur of doripenem is consistent with the hypothesis that the tautomeric state of the pyrroline ring contributes to the different carbapenem hydrolysis rates of OXA-1 and OXA-24. These findings represent a snapshot of a key step in the catalytic mechanism of an important class D enzyme, and might be useful for the design of novel inhibitors.
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Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70. Biochem J 2011; 432:495-504. [PMID: 21108605 DOI: 10.1042/bj20101122] [Citation(s) in RCA: 34] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl-enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.
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Pelto RB, Pratt RF. Serendipitous discovery of α-hydroxyalkyl esters as β-lactamase substrates. Biochemistry 2010; 49:10496-506. [PMID: 21087009 DOI: 10.1021/bi101071r] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
O-(1-Carboxy-1-alkyloxycarbonyl) hydroxamates were found to spontaneously decarboxylate in aqueous neutral buffer to form O-(2-hydroxyalkylcarbonyl) hydroxamates. While the former molecules do not react rapidly with serine β-lactamases, the latter are quite good substrates of representative class A and C, but not D, enzymes, and particularly of a class C enzyme. The enzymes catalyze hydrolysis of these compounds to a mixture of the α-hydroxy acid and hydroxamate. Analogous compounds containing aryloxy leaving groups rather that hydroxamates are also substrates. Structure-activity experiments showed that the α-hydroxyl group was required for any substantial substrate activity. Although both d- and l-α-hydroxy acid derivatives were substrates, the former were preferred. The response of the class C activity to pH and to alternative nucleophiles (methanol and d-phenylalanine) suggested that the same active site functional groups participated in catalysis as for classical substrates. Molecular modeling was employed to explore how the α-hydroxy group might interact with the class C β-lactamase active site. Incorporation of the α-hydroxyalkyl moiety into novel inhibitors will be of considerable interest.
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Affiliation(s)
- Ryan B Pelto
- Department of Chemistry, Wesleyan University, Middletown, Connecticut 06459, United States
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Bebrone C, Lassaux P, Vercheval L, Sohier JS, Jehaes A, Sauvage E, Galleni M. Current challenges in antimicrobial chemotherapy: focus on ß-lactamase inhibition. Drugs 2010; 70:651-79. [PMID: 20394454 DOI: 10.2165/11318430-000000000-00000] [Citation(s) in RCA: 120] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/02/2022]
Abstract
The use of the three classical beta-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam) in combination with beta-lactam antibacterials is currently the most successful strategy to combat beta-lactamase-mediated resistance. However, these inhibitors are efficient in inactivating only class A beta-lactamases and the efficiency of the inhibitor/antibacterial combination can be compromised by several mechanisms, such as the production of naturally resistant class B or class D enzymes, the hyperproduction of AmpC or even the production of evolved inhibitor-resistant class A enzymes. Thus, there is an urgent need for the development of novel inhibitors. For serine active enzymes (classes A, C and D), derivatives of the beta-lactam ring such as 6-beta-halogenopenicillanates, beta-lactam sulfones, penems and oxapenems, monobactams or trinems seem to be potential starting points to design efficient molecules (such as AM-112 and LK-157). Moreover, a promising non-beta-lactam molecule, NXL-104, is now under clinical development. In contrast, an ideal inhibitor of metallo-beta-lactamases (class B) remains to be found, despite the huge number of potential molecules already described (biphenyl tetrazoles, cysteinyl peptides, mercaptocarboxylates, succinic acid derivatives, etc.). The search for such an inhibitor is complicated by the absence of a covalent intermediate in their catalytic mechanisms and the fact that beta-lactam derivatives often behave as substrates rather than as inhibitors. Currently, the most promising broad-spectrum inhibitors of class B enzymes are molecules presenting chelating groups (thiols, carboxylates, etc.) combined with an aromatic group. This review describes all the types of molecules already tested as potential beta-lactamase inhibitors and thus constitutes an update of the current status in beta-lactamase inhibitor discovery.
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Affiliation(s)
- Carine Bebrone
- Biological Macromolecules, Centre for Protein Engineering, University of Liège, Liège, Belgium.
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40
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Li Y, Yu X, Ho J, Fushman D, Allewell NM, Tuchman M, Shi D. Reversible post-translational carboxylation modulates the enzymatic activity of N-acetyl-L-ornithine transcarbamylase. Biochemistry 2010; 49:6887-95. [PMID: 20695527 DOI: 10.1021/bi1007386] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/29/2022]
Abstract
N-Acetyl-l-ornithine transcarbamylase (AOTCase), rather than ornithine transcarbamylase (OTCase), is the essential carbamylase enzyme in the arginine biosynthesis of several plant and human pathogens. The specificity of this unique enzyme provides a potential target for controlling the spread of these pathogens. Recently, several crystal structures of AOTCase from Xanthomonas campestris (xc) have been determined. In these structures, an unexplained electron density at the tip of the Lys302 side chain was observed. Using (13)C NMR spectroscopy, we show herein that Lys302 is post-translationally carboxylated. The structure of wild-type AOTCase in a complex with the bisubstrate analogue N(delta)-(phosphonoacetyl)-N(alpha)-acetyl-l-ornithine (PALAO) indicates that the carboxyl group on Lys302 forms a strong hydrogen bonding network with surrounding active site residues, Lys252, Ser253, His293, and Glu92 from the adjacent subunit either directly or via a water molecule. Furthermore, the carboxyl group is involved in binding N-acetyl-l-ornithine via a water molecule. Activity assays with the wild-type enzyme and several mutants demonstrate that the post-translational modification of lysine 302 has an important role in catalysis.
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Affiliation(s)
- Yongdong Li
- Research Center for Genetic Medicine and Department of Integrative Systems Biology, Children's National Medical Center, The George Washington University, Washington, DC 20010, USA
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Schneider KD, Karpen ME, Bonomo RA, Leonard DA, Powers RA. The 1.4 A crystal structure of the class D beta-lactamase OXA-1 complexed with doripenem. Biochemistry 2010; 48:11840-7. [PMID: 19919101 DOI: 10.1021/bi901690r] [Citation(s) in RCA: 54] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/28/2022]
Abstract
The clinical efficacy of carbapenem antibiotics depends on their resistance to the hydrolytic action of beta-lactamase enzymes. The structure of the class D beta-lactamase OXA-1 as an acyl complex with the carbapenem doripenem was determined to 1.4 A resolution. Unlike most class A and class C carbapenem complexes, the acyl carbonyl oxygen in the OXA-1-doripenem complex is bound in the oxyanion hole. Interestingly, no water molecules were observed in the vicinity of the acyl linkage, providing an explanation for why carbapenems inhibit OXA-1. The side chain amine of K70 remains fully carboxylated in the acyl structure, and the resulting carbamate group forms a hydrogen bond to the alcohol of the 6alpha-hydroxyethyl moiety of doripenem. The carboxylate attached to the beta-lactam ring of doripenem is stabilized by a salt bridge to K212 and a hydrogen bond with T213, in lieu of the interaction with an arginine side chain found in most other beta-lactamase-beta-lactam complexes (e.g., R244 in the class A member TEM-1). This novel set of interactions with the carboxylate results in a major shift of the carbapenem's pyrroline ring compared to the structure of the same ring in meropenem bound to OXA-13. Additionally, bond angles of the pyrroline ring suggest that after acylation, doripenem adopts the Delta(1) tautomer. These findings provide important insights into the role that carbapenems may have in the inactivation process of class D beta-lactamases.
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Affiliation(s)
- Kyle D Schneider
- Department of Chemistry, Grand Valley State University, Allendale, Michigan 49401, USA
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Abstract
Since the introduction of penicillin, beta-lactam antibiotics have been the antimicrobial agents of choice. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial beta-lactamases. beta-Lactamases are now responsible for resistance to penicillins, extended-spectrum cephalosporins, monobactams, and carbapenems. In order to overcome beta-lactamase-mediated resistance, beta-lactamase inhibitors (clavulanate, sulbactam, and tazobactam) were introduced into clinical practice. These inhibitors greatly enhance the efficacy of their partner beta-lactams (amoxicillin, ampicillin, piperacillin, and ticarcillin) in the treatment of serious Enterobacteriaceae and penicillin-resistant staphylococcal infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to beta-lactam-beta-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant beta-lactamases from other classes that are resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of beta-lactams. Here, we review the catalytic mechanisms of each beta-lactamase class. We then discuss approaches for circumventing beta-lactamase-mediated resistance, including properties and characteristics of mechanism-based inactivators. We next highlight the mechanisms of action and salient clinical and microbiological features of beta-lactamase inhibitors. We also emphasize their therapeutic applications. We close by focusing on novel compounds and the chemical features of these agents that may contribute to a "second generation" of inhibitors. The goal for the next 3 decades will be to design inhibitors that will be effective for more than a single class of beta-lactamases.
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Affiliation(s)
- Sarah M. Drawz
- Departments of Pathology, Medicine, Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio
| | - Robert A. Bonomo
- Departments of Pathology, Medicine, Pharmacology, Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio, Research Service, Louis Stokes Cleveland Department of Veterans Affairs Medical Center, Cleveland, Ohio
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Baurin S, Vercheval L, Bouillenne F, Falzone C, Brans A, Jacquamet L, Ferrer JL, Sauvage E, Dehareng D, Frère JM, Charlier P, Galleni M, Kerff F. Critical role of tryptophan 154 for the activity and stability of class D beta-lactamases. Biochemistry 2009; 48:11252-63. [PMID: 19860471 DOI: 10.1021/bi901548c] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022]
Abstract
The catalytic efficiency of the class D beta-lactamase OXA-10 depends critically on an unusual carboxylated lysine as the general base residue for both the enzyme acylation and deacylation steps of catalysis. Evidence is presented that the interaction between the indole group of Trp154 and the carboxylated lysine is essential for the stability of the posttranslationally modified Lys70. Substitution of Trp154 by Gly, Ala, or Phe yielded noncarboxylated enzymes which displayed poor catalytic efficiencies and reduced stability when compared to the wild-type OXA-10. The W154H mutant was partially carboxylated. In addition, the maximum values of k(cat) and k(cat)/K(M) were shifted toward pH 7, indicating that the carboxylation state of Lys70 is dependent on the protonation level of the histidine. A comparison of the three-dimensional structures of the different proteins also indicated that the Trp154 mutations did not modify the overall structures of OXA-10 but induced an increased flexibility of the Omega-loop in the active site. Finally, the deacylation-impaired W154A mutant was used to determine the structure of the acyl-enzyme complex with benzylpenicillin. These results indicate a role of the Lys70 carboxylation during the deacylation step and emphasize the importance of Trp154 for the ideal positioning of active site residues leading to an optimum activity.
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Affiliation(s)
- Stéphane Baurin
- Laboratory of Biological Macromolecules, Center for Protein Engineering, University of Liège, Institut de Chimie B6a, Belgium
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