STRUCTURAL AND FUNCTIONAL ANALYSIS OF CARBOHYDRATE-TRANSFORMING AND GLYCOSIDATION ENZYMES IN THE MICROBIAL SECONDARY METABOLISM

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Well recognized importance of sugar transformation and glycosidation for the development of bioactive fine chemicals and industrial materials prompted us to study the enzymes involved in the biosynthesis of 2-deoxystreptamine-containing amino-glycoside antibiotics in the microbial secondary metabolism.The targeted glycosidation was the formation of paromamine, which is composed of D-glucosamine and 2-deoxystreptamine. We first established quantitative analytical methods of paromamine with HPLC and synthetic methods for NDP-D-glucosamine (N=A,T,G,C,U) as plausible glycosyl donor. Cell-free systems were prepared from Streptomyces fradiae, St.kanamyceticus, St.ribosidificus and Bacillus circulans by sonication, and incubations of each NDP-D-glucosamine in the presence of 2-deoxystreptamine with these cell-free preparations under various conditions have been carried out.2-Deoxystreptamine is a unique carbocycle biosynthesized by intramolecular cyclization of D-glucose via 2-deoxy-scyllo-in … More osose (DOI). We constructed a quantitative analytical method of the reaction with HPLC and GC-MS.A cell-free preparation derived fromSt. fradiae clearly showed the activity of converting D-glucose-6-phosphate (G-6-P) into DOI in the presence of NAD^+. The precise reaction mechanism was studied with a partially purified enzyme (named as DOI synthase) using D- [4-^2H,3-^<18>O] G-6-P synthesized by our own methodology. The reaction involving 1) oxidation at C-4 of G-6-P with NAD^+,2) elimination of phosphate from C-5 and C-6,3) in situ reduction of C-4 carbonyl with the enzyme-bound NADH,and 4) aldol reaction, is apparently pursued by a single DOI synthase. DOI synthase has also been isolated from Bacillus circulans and purification progressed. The estimated molecular weight is around 40K-60K.For the search of the DOI synthase gene, genomic DNA of B.circulans was partially digested with restriction enzymes and the resulting DNA fragments were cloned into E.coli plasmid pUC19. Each E.coli transformant was grown and tested for the activity of DOI synthase. So far, ca.1400 strains have been tested without success.The mechanism of a chemical version of DOI synthase, known as the Ferrier reaction, was analyzed by using stereoselective isotope-labeling and precise structural analysis of the reaction products and intermadiates. Involvement of a radical species has been proposed. Less