Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase

Biochemistry. 1999 Sep 7;38(36):11827-33. doi: 10.1021/bi991025h.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkylation
  • Amino Acid Sequence
  • Carbohydrate Sequence
  • Cloning, Molecular
  • Flavin-Adenine Dinucleotide / metabolism
  • Humans
  • Kinetics
  • Molecular Sequence Data
  • Mycobacterium tuberculosis / enzymology*
  • NADP / metabolism
  • Oxidation-Reduction
  • Oxidoreductases / genetics
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • Protein Conformation
  • Sequence Homology, Amino Acid
  • Spectrum Analysis


  • Flavin-Adenine Dinucleotide
  • NADP
  • Oxidoreductases
  • mycothione reductase