Expression, Purification, and Characterization of Mycobacterium tuberculosis Mycothione Reductase
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- 作者：Mehul P. Patel and John S. Blanchard
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：September 7, 1999
- 年：1999
- 卷：38
- 期：36
- 页码：11827 - 11833
- 全文大小：139K
- 年卷期：v.38,no.36(September 7, 1999)
- ISSN：1520-4995
文摘
Mycothione reductase from the human pathogen Mycobacterium tuberculosis has been cloned,expressed in Mycobacterium smegmatis, and purified 145-fold to homogeneity in 43% yield. Amino acidsequence alignment of mycothione reductase with the functionally homologous glutathione andtrypanothione reductase indicates conservation of the catalytically important redox-active disulfide,histidine-glutamate ion pair, and regions involved in binding both the FAD cofactor and the substrateNADPH. The homogeneous 50 kDa subunit enzyme exists as a homodimer and is NADPH-dependentand highly specific for the structurally unique low-molecular mass disulfide, mycothione, exhibitingMichaelis constants of 8 and 73 
M for NADPH and mycothione, respectively. HPLC analysis indicatedthe presence of 1 mol of bound FAD per monomer as the cofactor exhibiting an absorption spectrum witha 
max at 462 nm with an extinction coefficient of 11 300 M-1 cm-1. The reductive titration of the enzymewith NADH indicates the presence of a charge-transfer complex of one of the presumptive catalytic thiolatesand FAD absorbing at ca. 530 nm. Reaction with serially truncated mycothione and other disulfides andpyridine nucleotide analogues indicates a strict minimal disulfide substrate requirement for the glucosaminemoiety of mycothione. The enzyme exhibits bi-bi ping-pong kinetics with both disulfide and quinonesubstrates. Transhydrogenase activity is observed using NADH and thio-NADP+, confirming the kineticmechanism. We suggest mycothione reductase as the newest member of the class I flavoprotein disulfidereductase family of oxidoreductases.
M for NADPH and mycothione, respectively. HPLC analysis indicatedthe presence of 1 mol of bound FAD per monomer as the cofactor exhibiting an absorption spectrum witha max at 462 nm with an extinction coefficient of 11 300 M-1 cm-1. The reductive titration of the enzymewith NADH indicates the presence of a charge-transfer complex of one of the presumptive catalytic thiolatesand FAD absorbing at ca. 530 nm. Reaction with serially truncated mycothione and other disulfides andpyridine nucleotide analogues indicates a strict minimal disulfide substrate requirement for the glucosaminemoiety of mycothione. The enzyme exhibits bi-bi ping-pong kinetics with both disulfide and quinonesubstrates. Transhydrogenase activity is observed using NADH and thio-NADP+, confirming the kineticmechanism. We suggest mycothione reductase as the newest member of the class I flavoprotein disulfidereductase family of oxidoreductases.