Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Z. W. Chen; C. Y. Jiang; Qunxin She; S. J. Liu; P. J. Zhou

doi:10.1128/AEM.71.2.621-628.2005

Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Z. W. Chen, C. Y. Jiang, Qunxin She, S. J. Liu, P. J. Zhou

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

42 Citationer (Scopus)

Abstract

Udgivelsesdato: February 2005

Originalsprog	Engelsk
Tidsskrift	Applied and Environmental Microbiology
Vol/bind	71
Udgave nummer	2
Sider (fra-til)	621-628
ISSN	0099-2240
DOI	https://doi.org/10.1128/AEM.71.2.621-628.2005
Status	Udgivet - 2005

Adgang til dokumentet

10.1128/AEM.71.2.621-628.2005

Citationsformater

@article{d127de5074c211dbbee902004c4f4f50,

title = "Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis",

abstract = "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-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ 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, C31 and C101-X-X-C104, 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. ",

author = "Chen, {Z. W.} and Jiang, {C. Y.} and Qunxin She and Liu, {S. J.} and Zhou, {P. J.}",

year = "2005",

doi = "10.1128/AEM.71.2.621-628.2005",

language = "English",

volume = "71",

pages = "621--628",

journal = "Applied and Environmental Microbiology",

issn = "0099-2240",

publisher = "American Society for Microbiology",

number = "2",

}

TY - JOUR

T1 - Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

AU - Chen, Z. W.

AU - Jiang, C. Y.

AU - She, Qunxin

AU - Liu, S. J.

AU - Zhou, P. J.

PY - 2005

Y1 - 2005

N2 - 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-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ 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, C31 and C101-X-X-C104, 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.

AB - 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-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ 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, C31 and C101-X-X-C104, 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.

U2 - 10.1128/AEM.71.2.621-628.2005

DO - 10.1128/AEM.71.2.621-628.2005

M3 - Journal article

C2 - 15691910

SN - 0099-2240

VL - 71

SP - 621

EP - 628

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

IS - 2

ER -