Identification and characterization of 6-dehydroVB-A reductase from Streptomyces antibioticus

Streptomyces antibioticus NF-18 is a hyperproducing strain of a Streptomyce s hormone, virginiae butanolide A (VB-A), that induces virginiamycin produc tion of S. virginiae at nanomolar concentrations. To characterize the biosy nthetic pathway of VB-A, we identified and characterized for the first time the 6-dehydroVB-A reductase that is responsible for the final reduction st ep in the biosynthesis. Assay protocols and stabilization conditions were e stablished. The 6-dehydroVB-A reductase was found to require NADPH, not NAD H, as a coenzyme. The K-m values of the enzyme for NADPH and (+/-)-6-dehydr oVB-A were determined to be 50 +/- 2 mu M and 100 +/- 5 mu M, respectively. Ultracentrifugation experiments revealed that 6-dehydroVB-A reductase was present almost exclusively in the 100000 x g supernatant fraction, indicati ng that the enzyme is a cytoplasmic-soluble protein. The M-r of the native 6-dehydroVB-A reductase was estimated to be 82000 +/- 3000 by molecular sie ve HPLC. The optimal pH was found to be 6.7 +/- 0.2. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.