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

糖的转化和糖苷化反应在生物活性精细化学品和工业材料开发中的重要性已被广泛认识，这促使我们研究了微生物次级代谢中参与2-脱氧链霉胺氨基糖苷类抗生素生物合成的酶，目标糖苷化反应是由D-氨基葡萄糖和2-脱氧链霉胺组成的巴龙胺的形成。本论文首次建立了巴龙胺的HPLC定量分析方法和以NDP-D-氨基葡萄糖（N=A、T、G、C、U）为糖基供体的合成方法。本文报道了用超声法从草莓链霉菌、卡那霉素链霉菌、核糖化链霉菌和环状芽孢杆菌中制备无细胞体系，并在各种条件下，在2-脱氧链霉胺的存在下，将每种NDP-D-氨基葡萄糖与这些无细胞制备物一起孵育。2-脱氧链霉胺是一种独特的碳环化合物，通过2-脱氧-scylloin分子内环化D-葡萄糖合成 ...更多信息 蔗糖（DOI）。建立了一种高效液相色谱和气质联用的定量分析方法。弗氏菌在NAD^+存在下明显表现出将D-葡萄糖-6-磷酸（G-6-P）转化为DOI的活性。用我们自己的方法合成的D- [4-^2H，3-^ O] G-6-P，用部分纯化的酶（命名为DOI合成酶）研究了精确的反应机理<18>。反应包括1）G-6-P的C-4位被NAD^+氧化，2）C-5和C-6位的磷酸消除，3）C-4位羰基被酶结合的NADH原位还原，以及4）羟醛缩合反应，这些反应显然由单个DOI合酶进行。DOI合酶也已从环状芽孢杆菌中分离出来，并进行了纯化。为了寻找DOI合酶基因，用限制性内切酶部分消化环状B.circulans的基因组DNA，并将所得DNA片段克隆到大肠杆菌质粒pUC 19中。使每个大肠杆菌芽孢杆菌生长并测试DOI合酶的活性。到目前为止，约。1400株已测试没有successful.The机制的DOI合酶的化学版本，被称为Ferrier反应，进行了分析，通过使用立体选择性同位素标记和精确的结构分析的反应产物和intermadiates。已经提出了自由基物种的参与。少

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