42 アミノ配糖体抗生物質生合成における糖質環化鍵酵素2-デオキシ-scyllo-イノソース合成酵素の動的解析(口頭発表の部)

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2-Deoxy-scyllo-inosose (DOI) synthase is the key enzyme in the biosynthesis of aminocyclitol antibiotics and catalyzes cyclization of D-glucose-6-phosphate (G-6-P) into the 6-membered carbocycle DOI. DOI synthase from Bacillus circulans was isolated as a heterodimeric protein comprising from a 20kDa and a 40kDa subunits. While the 40kDa subunit (BtrC) is known to be responsible for catalysis, the function of the 20kDa subunit (BtrC2) has yet to be revealed. In this study, we first carried out identification of the gene encoding BtrC2 from B. circulans by reverse genetics. The amino acid sequence of BtrC2 deduced from btrC2 closely resembled to that of YaaE (function unknwon) of Bacillus subtilis. Instead, BtrC2 appeared to have sequence similarity to a certain extent with HisH of B. subtilis, an amidotransferase subunit of imidazole glycerol phosphate synthase. Disruption of btrC2 reduced the growth rate compared with the wild type, and simultaneously antibiotic producing activity was lost. Supplementation of NH_4Cl to the medium complemented only the growth rate of the disruptant, and both the growth rate and antibiotic production were restored by addition of yeast extract. These observations suggest that, in addition to the secondary metabolism, BtrC2 is mainly involved in the primary metabolism in B. circulans. In order to study the reaction mechanism and substrate-enzyme interaction of BtrC, a carbacyclic analog (C-6-P) was designed as a inhibitor. The synthesized C-6-P was found to have strong inhibitory activity with inhibition constant K_1, of 12μM. Furthermore, the C-6-P analog showed time- and concentration-dependent inactivation of BtrC (K_1=224μM, k_<inact>=3.22×10^<-3>s^<-1>). Subsequent LC/ESI-MS analysis of the enzyme after reaction with C-6-P showed the molecular mass of 40768, which was ca. 160mass unit larger than that of native BtrC (40608). These results clearly indicated that C-6-P was a mechanism-based irreversible inhibitor by a covalent bond formation. In order to precisely determine the amino acid residue binding to C-6-P, BtrC-C-6-P complex was digested by chymotrypsin, and the resulting peptide mixture was analyzed by LC/MS. From comparison of the peptides derived from native BtrC, C-6-P was found to bind to a 139-146 peptide fragment, SIKQAVNL, and further, Lys-141 was determined as a binding site of C-6-P by LC/MS/MS analysis. Lys-141 appears to recognize the phosphate group of the substrate G-6-P and play an important role in the DOI synthase reaction.
天然有機化合物討論会の論文
2002-09-01

42 アミノ配糖体抗生物質生合成における糖質環化鍵酵素2-デオキシ-scyllo-イノソース合成酵素の動的解析(口頭発表の部)

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