Key role of cysteine residues in catalysis and subcellular localization of sulfur oxygenase-reductase of Acidianus tengchongensis.
Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C(31) and C(101)-X-X-C(104); numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn(2+) strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C(31) and C(101)-X-X-C(104), in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.
Chen
Zhi-Wei
ZW
Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
Jiang
Cheng-Ying
CY
She
Qunxin
Q
Liu
Shuang-Jiang
SJ
Zhou
Pei-Jin
PJ
D041541:Acidianus / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D003545:Cysteine; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019848:Gene Expression Regulation, Archaeal; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D013347:Subcellular Fractions / Q000201:enzymology*; D013455:Sulfur / Q000378:metabolism
D019843:Archaeal Proteins; D013455:Sulfur; D010088:Oxidoreductases; D003545:Cysteine