Triple hydroxylation of tetracenomycin A2 to tetracenomycin C in Streptomyces glaucescens. Overexpression of the tcmG gene in Streptomyces lividans and characterization of the tetracenomycin A2 oxygenase

J Biol Chem. 1994 Dec 2;269(48):30726-33.


Nucleotide sequence analysis of the tcmG gene has suggested that the TcmG protein is responsible for the triple-hydroxylation of tetracenomycin (Tcm) A2 to Tcm C in Streptomyces glaucescens (Decker, H., Motamedi, H., and Hutchinson, C.R. (1993) J. Bacteriol. 175, 3876-3886). The heterologous expression of the tcmG gene in Streptomyces lividans and the purification and characterization of TcmG protein, which we have named Tcm A2 oxygenase, are described here. NH2-terminal amino acid analysis of the purified enzyme led to the revision of the translational start site of tcmG to a TTG, 33 base pairs downstream of the GTG site assigned initially on the basis of nucleotide sequence analysis. Tcm A2 oxygenase is a monomeric protein in solution and contains 1 mol of non-covalently bound FAD; the apoenzyme can be partially reconstituted in vitro by addition of FAD. Tcm A2 oxygenase exhibits an optimal pH of 9.0-9.5 and prefers NADPH over NADH as an electron donor. The apparent K'm of the enzyme for Tcm A2, NADH, and NADPH are 1.81 +/- 0.38, 260 +/- 19, and 82.1 +/- 17 microM, respectively, and the apparent V'max for the reaction is 14.7 +/- 1.1 nmol Tcm C/ Purification and characterization of Tcm A2 oxygenase provide direct evidence to support the notion that the angular hydroxy groups of naphthacenequinones like Tcm C are introduced from 18O2 via a mono- or dioxygenase process.

Publication types

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

MeSH terms

  • Amino Acid Sequence
  • Anthracyclines*
  • Anti-Bacterial Agents / biosynthesis
  • Anti-Bacterial Agents / metabolism*
  • Antibiotics, Antineoplastic / metabolism*
  • Apoenzymes / metabolism
  • Bacterial Proteins*
  • Base Sequence
  • Chromatography, Gel
  • Chromatography, Ion Exchange
  • DNA Primers
  • DNA, Bacterial / isolation & purification
  • DNA, Bacterial / metabolism
  • Escherichia coli
  • Gene Expression
  • Genes, Bacterial*
  • Hydroxylation
  • Molecular Sequence Data
  • NAD / metabolism
  • Naphthacenes / isolation & purification
  • Naphthacenes / metabolism
  • Oxygenases / biosynthesis
  • Oxygenases / isolation & purification
  • Oxygenases / metabolism*
  • Plasmids
  • Streptomyces / genetics
  • Streptomyces / metabolism*


  • Anthracyclines
  • Anti-Bacterial Agents
  • Antibiotics, Antineoplastic
  • Apoenzymes
  • Bacterial Proteins
  • DNA Primers
  • DNA, Bacterial
  • Naphthacenes
  • NAD
  • tetracenomycin C
  • tetracenomycin A2
  • Oxygenases
  • TcmH protein, Streptomyces glaucescens

Associated data

  • GENBANK/M80674