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.
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.
Shen
B
B
School of Pharmacy, Madison, WI 53706.
Hutchinson
C R
CR
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*
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