Production of novel biologically active molecules based on the functional analysis of glycosylation enzymes related to secondary metabolism

本研究对链霉菌生产的c -糖基化抗生素的生物合成酶进行了功能分析，目标化合物是一类芳香抗生素苯并异铬马醌（BIQs）。代表是甲德霉素和granaticin，两者都携带独特的脱氧糖成分，这是很大的药学兴趣。本项目取得的成果如下：(1)甲德霉素生物合成过程中脱氧糖生物合成酶的功能表达。Med-17和med - 18酶在大肠杆菌中异种表达，这两个酶都与甲德霉素中氨甘胺成分中核苷酸-糖中间体的形成有关，并对其进行了功能表征。Med-17和med - 18分别与tdp -葡萄糖合成酶和tdp -葡萄糖4,6-脱水酶有关。(2) BIQ分子所涉及的还原酶和氧化酶的功能表征。放线菌素（ACT）是一种芳香聚酮类抗生素，由链霉菌colicolor A3(2)产生。在ACT生物合成中，立体定向酮还原酶（RED1）在大肠杆菌中异种表达，并与合成类似物对RED1进行酶学研究。该酶倾向于接受游离酸形式作为底物，这强烈表明RED1识别无蛋白酮酸作为后期生物合成步骤的第一个承诺中间体。另外，在参与ACT生物合成的actva -5和6基因的缺失突变体前分离出了一种新的分流产物actin - operylone。光谱分析显示其具有四个手性中心的苝酰醌型骨架，显然是由ACT中间体的二聚化而来。actinoperylone的结构表明ActVA-ORF5在C-6的氧引入中起重要作用。

This research deals with the functional analysis of the biosynthetic enzymes of C-glycosylated antibiotics produced by Streptomyces spp. The target compounds are a class of aromatic antibiotics known as benzoisochromanequinones (BIQs). Representative are medermycin and granaticin, both carry the unique deoxysugar components, which are of great pharmaceutical interest. The achievements in this project are as follows.(1) The functional expression of the deoxysugar biosynthetic enzymes involved in medermycin biosynthesis.The Med-17 and 18 enzymes, both related to the formation of nucleotide-sugar intermediates in angolosamine component in medermycin, are heterologously expressed in Escherichia coli, and the recombinant enzymes were functionally characterized. Med-17 and 18 were revealed to TDP-glucose synthase and TDP-glucose 4,6-dehydratase, respectively.(2) The functional characterization of the reducing and oxidizing enzymes involved in the BIQ molecules.Actinorhodin(ACT) produced by Streptomyces coelicolor A3(2) is an aromatic polyketide antibiotic. n the ACT biosynthesis, a stereospecific ketoreductase(RED1) was heterologously expressed in E. coli, and enzymatic studies on RED1 with synthetic analogues. The enzyme preferred to accept an free acid form as its substrate, strongly suggesting that RED1 recognizes a protein free keto acid as the first committed intermediate of later biosynthetic steps. Separately, a novel shunt product, actinoperylone, has been isolated front a deletion mutant of the actVA-5and 6 genes involved for ACT biosynthesis. Spectoroscopic analysis revealed its perylenquinone-type skeleton with the four chiral centers, obviously derived from the dimerization of an ACT intermediate. The structure of actinoperylone indicates the essential role of ActVA-ORF5 in the oxygen introduction at C-6.