Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones c alled 'butyrolactone autoregulators' which regulate the antibiotic producti on in Streptomyces species at nanomolar concentrations. Cell-free extract o f a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catal yzing the final step of the autoregulator biosynthesis, namely, the NADPH-d ependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type i somer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S )-enantiomers] were also produced from (+/-)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselecti vities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chir al HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyz ed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratio s of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antib ioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much fav ored toward VB-A biosynthesis, the other with relaxed stereoselectivity cap able of synthesizing both VB-type and IM-2-type autoregulators. (C) 2000 El sevier Science B.V. All rights reserved.