title: Site-specific mutagenesis and functional analysis of active sites of sulfur oxygenase reductase from Gram-positive moderate thermophile Sulfobacillus acidophilus TPY.
abstract: Sequence alignments revealed that the conserved motifs of SORSa 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 SORSa, cysteines 31, 101 and 104 (C31, C101, C104), histidines 86 and 90 (H86 and H90) and glutamate 114 (E114) of SORSa 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 SORSa 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.
authors.affiliation: School of Resource Processing and Bioengineering, Central South University, Changsha, Hunan 410083, People's Republic of China; Key Laboratory of Marine Biogenetic Resources, Third Institute of Oceanography, State Oceanic Administration, Xiamen, Fujian, China.
authors.forename: Huijun
authors.initials: H
authors.lastname: Zhang
authors.forename: Wenbin
authors.initials: W
authors.lastname: Guo
authors.forename: Changan
authors.initials: C
authors.lastname: Xu
authors.forename: Hongbo
authors.initials: H
authors.lastname: Zhou
authors.forename: Xinhua
authors.initials: X
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D020134:Catalytic Domain; D004252:DNA Mutational Analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006094:Gram-Positive Bacteria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D050862:Oxidoreductases Acting on Sulfur Group Donors
keywords: Enzyme activity; SOR(Aa); SOR(At); SOR(Hn); SOR(Sa); Site-specific mutagenesis; Sulfobacillus acidophilus TPY; Sulfur oxygenase reductase; the SOR of Acidianus ambivalens; the SOR of Acidianus tengchongensis; the SOR of Halothiobacillus neapolitanus; the SOR of Sulfobacillus acidophilus TPY