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 acid sequence alignment of mycothione reductase with the functionally homologous glutathione and trypanothione 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 substrate NADPH. The homogeneous 50 kDa subunit enzyme exists as a homodimer and is NADPH-dependent and highly specific for the structurally unique low-molecular mass disulfide, mycothione, exhibiting Michaelis constants of 8 and 73 mM for NADPH and mycothione, respectively. HPLC analysis indicated the presence of 1 mol of bound FAD per monomer as the cofactor exhibiting an absorption spectrum with a λ(max) at 462 nm with an extinction coefficient of 11 300 M-1 cm-1. The reductive titration of the enzyme with NADH indicates the presence of a charge-transfer complex of one of the presumptive catalytic thiolates and FAD absorbing at ca. 530 nm. Reaction with serially truncated mycothione and other disulfides and pyridine nucleotide analogues indicates a strict minimal disulfide substrate requirement for the glucosamine moiety of mycothione. The enzyme exhibits bi- bi ping-pong kinetics with both disulfide and quinone substrates. Transhydrogenase activity is observed using NADH and thio-NADP+, confirming the kinetic mechanism. We suggest mycothione reductase as the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.
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