Basic Study
Copyright ©The Author(s) 2015. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Biol Chem. Aug 26, 2015; 6(3): 249-264
Published online Aug 26, 2015. doi: 10.4331/wjbc.v6.i3.249
Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis
Mee-Kyung Cha, Yoo-Jeen Bae, Kyu-Jeong Kim, Byung-Joon Park, Il-Han Kim
Mee-Kyung Cha, Yoo-Jeen Bae, Kyu-Jeong Kim, Byung-Joon Park, Il-Han Kim, Department of Life Science and Technology, Daeduk Valley Campus, Paichai University, Taejon 305-509, South Korea
Author contributions: Cha MK was involved in acquisition of data and interpretation; Kim IH was involved in design of the study and drafting the manuscript; Bae YJ, Kim KJ and Park BJ performed the experiments throughout this work; all authors approved this version of the manuscript to be published.
Supported by The Basic Science Research Program through the Korea Research Foundation Grant funded by the Ministry of Education, Science, and Technology (NRF-2011-0008913); Kim IH and Cha MK performed this work during their research sabbatical supported by Paichai University (2014-2015).
Institutional review board statement: This article is not describing a study involving human and/or animal subjects. Therefore IRB statement is not required.
Conflict-of-interest statement: The authors report no declarations of interest.
Data sharing statement: It is an open access article.
Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Correspondence to: Il-Han Kim, PhD, Department of Life Science and Technology, Daeduk Valley Campus, Paichai University, 11-3 Techno 1-ro, Daejeon, Taejon 305-509, South Korea. ihkim@pcu.ac.kr
Telephone: +82-42-5205379
Received: February 2, 2015
Peer-review started: February 4, 2015
First decision: April 10, 2015
Revised: May 21, 2015
Accepted: June 9, 2015
Article in press: June 11, 2015
Published online: August 26, 2015
Processing time: 204 Days and 23.2 Hours
Abstract

AIM: To identify alkyl hydroperoxide reductase subunit C (AhpC) homologs in Bacillus subtilis (B. subtilis) and to characterize their structural and biochemical properties. AhpC is responsible for the detoxification of reactive oxygen species in bacteria.

METHODS: Two AhpC homologs (AhpC_H1 and AhpC_H2) were identified by searching the B. subtilis database; these were then cloned and expressed in Escherichia coli. AhpC mutants carrying substitutions of catalytically important Cys residues (C37S, C47S, C166S, C37/47S, C37/166S, C47/166S, and C37/47/166S for AhpC_H1; C52S, C169S, and C52/169S for AhpC_H2) were obtained by site-directed mutagenesis and purified, and their structure-function relationship was analyzed. The B. subtilis ahpC genes were disrupted by the short flanking homology method, and the phenotypes of the resulting AhpC-deficient bacteria were examined.

RESULTS: Comparative characterization of AhpC homologs indicates that AhpC_H1 contains an extra C37, which forms a disulfide bond with the peroxidatic C47, and behaves like an atypical 2-Cys AhpC, while AhpC_H2 functions like a typical 2-Cys AhpC. Tryptic digestion analysis demonstrated the presence of intramolecular Cys37-Cys47 linkage, which could be reduced by thioredoxin, resulting in the association of the dimer into higher-molecular-mass complexes. Peroxidase activity analysis of Cys→Ser mutants indicated that three Cys residues were involved in the catalysis. AhpC_H1 was resistant to inactivation by peroxide substrates, but had lower activity at physiological H2O2 concentrations compared to AhpC_H2, suggesting that in B. subtilis, the enzymes may be physiologically functional at different substrate concentrations. The exposure to organic peroxides induced AhpC_H1 expression, while AhpC_H1-deficient mutants exhibited growth retardation in the stationary phase, suggesting the role of AhpC_H1 as an antioxidant scavenger of lipid hydroperoxides and a stress-response factor in B. subtilis.

CONCLUSION: AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.

Keywords: Cysteine-dependent peroxidase; Thioredoxin; Thiol peroxidase; Peroxiredoxin; Alkyl hydroperoxide; Ortholog; Bacillus subtilis; Oxidative stress

Core tip: Two alkyl hydroperoxide reductase subunit C (AhpC) homologs (AhpC_H1 and AhpC_H2) were identified by searching the Bacillus subtilis database. Sequence homology and phylogenetic analyses revealed that AhpC_H1 is an ortholog of Escherichia coli (E. coli) AhpC, a representative of bacterial AhpC. AhpC_H1 forms dimers consisting of atypical 2-Cys subunits, while AhpC_H2 behaves like a typical 2-Cys AhpC. These AhpC homologs may perform their respective physiological functions at different peroxide levels. Structural and catalytic differences between the enzymes indicate that AhpC_H1 is not an ortholog of E. coli AhpC, but a novel type of atypical 2-Cys AhpC.