Biosynthesis of nanaomycin. II. Purification and properties of nanaomycin D reductase involved in the formation of nanaomycin A from nanaomycin D1.
Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D leads to A leads to E leads to B) in Streptomyces rosa var. notoensis, was purified from the crude extract of the strain by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, Sephadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a flavoprotein which contains FAD as a prosthetic group and has a molecular weight of 68,000 daltons. It catalyzed the reductive transformation of nanaomycin D to nanaomycin A in the presence of NADH under anaerobic conditions. The Km values were 250 microM for nanaomycin D and 62 microM for NADH. The enzyme was inhibited by 1 mM Cu2+ ion and by NADH at concentrations over 50 microM. The optimal pH was 5.0 and the optimal temperature was 37 degrees C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and frenolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4 alpha, 10 alpha-epoxynanaomycin D were not converted.
D000900:Anti-Bacterial Agents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D009285:Naphthoquinones / Q000096:biosynthesis / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013057:Spectrum Analysis; D013302:Streptomyces / Q000201:enzymology*; D013379:Substrate Specificity; D013696:Temperature; D000095622:Benzoisochromanequinones
D000900:Anti-Bacterial Agents; D009243:NAD; D009285:Naphthoquinones; D010088:Oxidoreductases; C008638:nanaomycin A; D000095622:Benzoisochromanequinones; C013079:kalafungin; C032455:nanaomycin D reductase