33 2-デオキシストレプタミン含有抗生物質生合成系の分子解析(口頭発表の部)

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The biosynthesis of clinically important aminocyclitol antibiotics, particularly containing 2-deoxystreptamine, has been studied. The first-step enzyme, 2-deoxy-scyllo-inosose synthase (DOIS), was purified to homogeneity from butirosin-producing Bacillus circulans for the first time as heterodimer consisting of 23kDa and 41kDa subunits. Partial sequences of the polypeptides were deduced and reverse-genetics approach consisting of oligonucleotide probe and hybridization allowed identifying the responsible gene btrC for the larger subunit. The deduced amino acid sequence appeared to have significant homology with microbial dehydroquinate synthase (DHQS). The btrC gene was heterologously over-expressed in E. coli and the expressed BtrC was recovered by rather simple purification procedures. Mechanistic studies showed that the stereochemistry of Bacillus DOIS was identical with that of Streptomyces DOIS, but different from DHQS. The possible DOIS-substrate interaction has been postulated on the basis of the studies with substrate analogs and molecular modeling. The butirosin biosynthetic gene cluster was analyzed by gene-walking starting from btrC, and more than a dozen ORFs were identified. Gene disruption of the ORFs, btrB, btrC, btrD, and btrM, were carried out and clearly showed that every disruptants lost the ability of antibiotic production. Therefore, the above-mentioned gene cluster, which has not yet been fully deduced, appeared to be responsible for the biosynthesis of butirosin. The function of each ORF has further been discussed by homology search.
天然有機化合物討論会の論文
2000-10-01

33 2-デオキシストレプタミン含有抗生物質生合成系の分子解析(口頭発表の部)

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