title: 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.
abstract: 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/min.mg. 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.
authors.affiliation: School of Pharmacy, Madison, WI 53706.
authors.forename: B
authors.initials: B
authors.lastname: Shen
authors.forename: C R
authors.initials: CR
authors.lastname: Hutchinson
mesh_terms: D000595:Amino Acid Sequence; D018943:Anthracyclines; D000900:Anti-Bacterial Agents / Q000096:biosynthesis / Q000378:metabolism*; D000903:Antibiotics, Antineoplastic / Q000378:metabolism*; D001051:Apoenzymes / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D006900:Hydroxylation; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D009279:Naphthacenes / Q000302:isolation & purification / Q000378:metabolism; D010105:Oxygenases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism*
chemical_list: D018943:Anthracyclines; D000900:Anti-Bacterial Agents; D000903:Antibiotics, Antineoplastic; D001051:Apoenzymes; D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D009279:Naphthacenes; D009243:NAD; C020938:tetracenomycin C; C090681:tetracenomycin A2; D010105:Oxygenases; C082096:TcmH protein, Streptomyces glaucescens