Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY

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• 作者：Huijun Zhang ; Wenbin Guo ; Changan Xu ; Hongbo Zhou ; Xinhua Chen
• 关键词：SORSa ; the SOR of Sulfobacillus acidophilus TPY ; SORAa ; the SOR of Acidianus ambivalens ; SORAt ; the SOR of Acidianus tengchongensis ; SORHn ; the SOR of Halothiobacillus neapolitanus
• 刊名：Microbiological Research
• 出版年：2013
• 出版时间：14 December, 2013
• 年：2013
• 卷：168
• 期：10
• 页码：654-660
• 全文大小：4892 K

文摘

Sequence alignments revealed that the conserved motifs of SOR_Sa which formed an independent branch between archaea and Gram-negative bacteria SORs according to the phylogenetic relationship were similar with the archaea and Gram-negative bacteria SORs. In order to investigate the active sites of SOR_Sa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SOR_Sa were chosen as the target amino acid residues for site-specific mutagenesis. The wild type and six mutant SORs were expressed in E. coli BL21, purified and confirmed by SDS-PAGE and Western blotting analysis. Enzyme activity determination revealed that the active sites of SOR_Sa were identical with the archaea and Gram-negative bacteria SORs reported. Replacement of any cysteine residues reduced SOR activity by 53-100 % , while the mutants of H86A, H90A and E114A lost their enzyme activities largely, only remaining 20 % , 19 % and 32 % activity of the wild type SOR respectively. This study will enrich our awareness for active sites of SOR in a Gram-positive bacterium.