Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase.
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 microM 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 lambda(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.
Patel
M P
MP
Department of Biochemistry, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York 10461, USA.
Blanchard
J S
JS
D000478:Alkylation; D000595:Amino Acid Sequence; D002240:Carbohydrate Sequence; D003001:Cloning, Molecular; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013057:Spectrum Analysis
D005182:Flavin-Adenine Dinucleotide; D009249:NADP; D010088:Oxidoreductases; C121837:mycothione reductase