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

微生物二次代谢中碳水化合物转化酶和糖苷化酶的结构和功能分析

• 批准号: 05453202
• 负责人: KAKINUMA Katsumi
• 金额: $ 4.8万
• 依托单位: TOKYO INSTITUTE OF TECHNOLOGY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1993
• 资助国家: 日本
• 起止时间: 1993 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-05453202/
• 关键词: MICROBIAL SECONDARY METABOLISM; AMINOGLYCOSIDE ANTIBIOTICS; BIOSYNTHESIS; PAROMAMINE FORMATION; 2-DEOXY-SCYLLO-INOSOSE; 2-DEOXY-SCYLLO-INOSOSE SYNTHASE; D- [4-^2H,3-^<18>O] GLUCOSE; FERRIER REACTION; NAD結合性酵素; 二重標識; [4-^2H,3-^<18>O]グルコース-6ーリン酸 /

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

人们认识到糖转化和糖化对生物活性精细化学物质和工业材料的发展的重要性，促使我们研究涉及2-脱氧氨基氨基氨基氨基糖苷的生物合成的酶2-脱氧抑制剂。我们首先用HPLC和合成方法建立了NDP-D-葡萄糖胺（N = A，T，G，C，U）的定量分析方法，作为合理的糖基供体。从链霉菌，圣卡纳木毒素，圣烷瘤，圣里伯氏菌和芽孢杆菌通过的无单细胞系统制备，并通过在各种条件下与这些cell型的不同条件携带的是2-脱氧蛋白毒素的每种NDP-D-D-D-葡萄糖胺在存在的2-脱氧蛋白质的情况下进行。 D-葡萄糖通过2-脱氧 - cyllo-In…更多的欧司糖（DOI）的环化。我们构建了一种与HPLC和GC-MS.一种无细胞制剂的反应的定量分析方法。 Fradiae清楚地表明，在NAD^+存在下，将D-葡萄糖-6-磷酸（G-6-P）转化为DOI的活性。使用D- [4-^2H，3 - ^<18> o] G-6-P合成通过我们自己的方法，使用D- [4-^2H，3 - ^<18> O] G-6-P研究了精度反应机理（称为DOI合酶）。涉及1）G-6-P在C-4的反应与NAD^+，2）从C-5和C-6,3）消除C-4 Carbonyl与酶结合的NADH和4）Aldol反应的原位降低，显然是由单个DOII-DOII合酶追求的。 DOI合酶也已从芽孢杆菌电路中分离出来，并进行了纯化。估计的分子量约为40K-60K。对于搜索DOI合酶基因，b.circulans的基因组DNA被部分消化，并用限制酶消化，并将所得的DNA片段克隆到E.coli质粒PUC19中。每种大肠杆菌转化剂均可生长并测试DOI合酶的活性。到目前为止，已经对CA.1400菌株进行了测试，但没有成功。通过使用立体选择性同位素标记的DOI合酶（称为Ferrier反应）的化学形式的机理，对反应产物和中间体的精确结构分析进行了分析。已经提出了激进物种的参与。较少的

项目成果

N.YAMAUCHI AND K.KAKINUMA: "BIOCHEMICAL STUDIES ON 2-DEOXY-SCYLLO-INOSOSE,AN EARLY INTERMEDIATE IN THE BIOSYNTHESIS OF 2-DEOXYSTREPTAMINE.IV.A CLUE TO THE MECHANISTIC SIMILARITY OF 2-DEOXY-SCYLLO-INOSOSE SYNTHASE IN THE 2-DEOXYSTREPTAMINE BIOSYNTHESIS TO

N.Yamauchi 和 K.Kakinuma：“2-脱氧青蟹肌糖的生物化学研究，2-脱氧链霉胺生物合成的早期中间体。IV.2-脱氧青蟹肌糖合成酶在

Noriaki Yamauchi: "Mechanistic and stereochemical studies on Ferrier reaction by means of chirally deuterated glucose" Tetrahedron. 50. 4125-4136 (1994)

Noriaki Yamauchi：“通过手性氘代葡萄糖进行费里尔反应的机理和立体化学研究”四面体。

Y.TOYOOKA,T.MATSUZAWA,T.EGUCHI,AND K.KAKINUMA: "NADH MIMICS ON DIACETONE-D-GLUCOSE : STEREOSELECTIVE BIOMIMETIC REDUCTION OF BENZOYLFORMATE AND INTERPRETATION OF CHIRALITY TRANSFER DEDUCED BY MOLECULAR ORBITAL APPROACH." TETRAHEDRON. 51 [23]. 6459-6474 (1

Y.TOYOOKA、T.MATSUZAWA、T.EGUCHI 和 K.KAKINUMA：“双丙酮-D-葡萄糖上的 NADH 模拟物：苯甲酰甲酸的立体选择性仿生还原以及分子轨道方法推导的手性传递的解释。”

Noriaki Yamauchi: "Mechanistic and stereochenical studies on Ferrier reaction by means of chirally deuterated glucose" Tetrahedron. 50. 4125-4136 (1994)

Noriaki Yamauchi：“通过手性氘化葡萄糖进行费里尔反应的机理和立体化学研究”四面体。

Noriaki Yamauchi: "Biochemical studies on 2-deoxy-suyllo-inosore,an early informediotc in the biosynthesis of 2-deoxystreptamine.TV.A due to the mechamistic similartty of 2-deoxy-scyllo-inosose symthase in the 2-deoxy-streptamine biosyn thesrs to debydroq

Noriaki Yamauchi：“2-脱氧青蟹肌糖的生化研究，2-脱氧链霉胺生物合成中的一种早期信息。TV.A，由于 2-脱氧链霉胺中 2-脱氧青蟹肌糖合酶的机制相似