Cha Mee-Kyung, Bae Yoo-Jeen, Kim Kyu-Jeong, Park Byung-Joon, Kim Il-Han
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.
World J Biol Chem. 2015 Aug 26;6(3):249-64. doi: 10.4331/wjbc.v6.i3.249.
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.
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.
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.
AhpC_H1, a novel atypical 2-Cys AhpC, is functionally distinct from AhpC_H2, a typical 2-Cys AhpC.
鉴定枯草芽孢杆菌中的烷基过氧化氢还原酶亚基C（AhpC）同源物，并表征其结构和生化特性。AhpC负责细菌中活性氧的解毒。
通过搜索枯草芽孢杆菌数据库鉴定出两个AhpC同源物（AhpC_H1和AhpC_H2）；然后将它们克隆并在大肠杆菌中表达。通过定点诱变获得携带催化重要的半胱氨酸残基替代的AhpC突变体（AhpC_H1的C37S、C47S、C166S、C37/47S、C37/166S、C47/166S和C37/47/166S；AhpC_H2的C52S、C169S和C52/169S）并进行纯化，分析它们的结构-功能关系。通过短侧翼同源性方法破坏枯草芽孢杆菌的ahpC基因，并检查所得AhpC缺陷型细菌的表型。
AhpC同源物的比较表征表明，AhpC_H1含有一个额外的C37，它与过氧化物酶活性中心的C47形成二硫键，表现得像一个非典型的2-半胱氨酸AhpC，而AhpC_H2的功能像一个典型的2-半胱氨酸AhpC。胰蛋白酶消化分析表明存在分子内Cys37-Cys47连接，其可被硫氧还蛋白还原，导致二聚体缔合形成更高分子量的复合物。Cys→Ser突变体的过氧化物酶活性分析表明三个半胱氨酸残基参与催化。AhpC_H1对过氧化物底物的失活具有抗性，但在生理H2O2浓度下与AhpC_H2相比活性较低，这表明在枯草芽孢杆菌中，这些酶可能在不同底物浓度下具有生理功能。暴露于有机过氧化物会诱导AhpC_H1表达，而AhpC_H1缺陷型突变体在稳定期表现出生长迟缓，这表明AhpC_H1在枯草芽孢杆菌中作为脂质氢过氧化物的抗氧化清除剂和应激反应因子的作用。
AhpC_H1是一种新型非典型2-半胱氨酸AhpC，在功能上与典型的2-半胱氨酸AhpC_H
