title: Characterization of two alkyl hydroperoxide reductase C homologs alkyl hydroperoxide reductase C_H1 and alkyl hydroperoxide reductase C_H2 in Bacillus subtilis.
abstract: OBJECTIVE
  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.
  
  
  CONCLUSIONS
  AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.
authors: 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.
authors: Mee-Kyung
authors: MK
authors: Cha
authors: 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.
authors: Yoo-Jeen
authors: YJ
authors: Bae
authors: 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.
authors: Kyu-Jeong
authors: KJ
authors: Kim
authors: 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.
authors: Byung-Joon
authors: BJ
authors: Park
authors: 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.
authors: Il-Han
authors: IH
authors: Kim
keywords: Alkyl hydroperoxide; Bacillus subtilis; Cysteine-dependent peroxidase; Ortholog; Oxidative stress; Peroxiredoxin; Thiol peroxidase; Thioredoxin