尾仲 宏康

Staurosporine and rebeccamycin produced by actinomycetes are members of the family of indolocarbazole alkaloids that are expected to be the lead compounds of antitumor drugs, because they are strong inhibitors of Protein Kinase C or DNA topoisomerases, which play important role for cell cycling. Staurosporine biosynthetic gene cluster cloned from Streptomyces sp. TP-A0274 consists of 15 orfs spanning 22 kb, and rebeccamycin biosynthetic gene cluster cloned from Lechevarieria aerocolonigenes ATCC39243 consists of 10 orfs spanning 16 kb. Gene disruption and bioconversion experiments revealed the indolocarbazole biosynthetic pathway. First, indole-3-pyruvic acid, which is derived from tryptophan, is coupled by StaD, which is a over 500kDa large heme protein complex, to yield chromopyrrolic acid (CPA). Next, this dicarboxylic bisindole compound, CPA is transformed into the indolocarbazole skeleton via intramolecular C-C bond formation and oxidative decarboxylation catalyzed by cytochrome P450 StaP (StaP, CYP245A1). In this study, we also report X-ray crystal structures of CPA-bound and -free forms of StaP. Based on the crystal structure of StaP-CPA complex, we propose that C-C bond formation occurs via an indole cation radical intermediate. Our crystallographic study shows the first crystal structures of enzymes involved in formation of the indolocarbazole core and provides valuable insights into the process of indolocarbazole biosynthesis, combinatorial biosynthesis of indolocarbazoles, and the diversity of cytochrome P450 chemistry.

我々は、富山湾内にある採水施設で汲み上げられた深層水から、フィルター集菌し、フィルターをアジ化ナトリウム、シクロヘキシミド添加MRS寒天培地上に置床・培養することにより、増菌培養を行うことなく乳酸菌の分離を行った。この方法で、45lの深層水から25株の乳酸菌を分離した。得られた分離株から形態学的試験、生理学的試験および同定キットAPI20strepを用いて、Enterococcus faecium 8株、Enterococcus durans 2株およびEnterococcus faecalis 1株を選抜した。これらの菌株の内、E. faecium TN-3およびE. faecalis TN-9と判定された2株の16S rDNA解析では、TN-3 株はE. durans に、TN-9 株はE. faecalis に最も高い相同性を示した。また、これら2株はE. durans およびE. faecalis の基準株にそれぞれ同等の薬剤感受性を示した。TN-3 株、TN-9 株のvancomycinに対するMIC値はともに0.5μg/ml および1.0μg/ml であり、両菌株ともにvancomycin耐性遺伝子も検出されなかった。