Biosynthesis of Nanaomycin. II. Purification and Properties of Nanaomycin D Reductase Involved in the Formation of Nanaomycin A from Nanaomycin D1

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Abstract
Nanaomycin D reductase, catalyzing the conversion of nanaomycin D to nanaomycin A, which is the first step in the biosynthetic sequence (D←A←E←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, Se-phadex G-100 and hydroxyapatite to give an electrophoretically homogeneous preparation. The enzyme was found to be a fiavoprotein 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 μM for nanaomycin D and 62 μM for NADH. The enzyme was inhibited by 1 mM Cu2+ ion and by NADH at concentrations over 50 μM. The optimal pH was 5.0 and the optimal temperature was 37°C. Several benzoisochromane-quinone antibiotics other than nanaomycin D, kalafungin (enantiomer of nanaomycin D), griseucin A and fre-nolicin B were converted to the corresponding reduced products by the enzyme. However, granaticin and 4a,10a-epoxynanaomycin D were not converted.