Hiroyasu Onaka

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.

Twenty-five strains of lactic acid bacteria which were acid producing, catalase-negative and gram-positive had been isolated from 45l of deep-seawater samples in Toyama Bay, Japan by the membrane filter method performed using the modified de Man-Rogosa-Sharpe (MRS) agar medium. Eleven strains out of them were classified as 8 of Enterococcus faecium, as 2 of Enterococcus durans and as one of Enterococcus faecalis based on morphological and physiological characteristics, together with the results of API20strep identification kit. The strains TN-3 and TN-9, which were resembled E. faecium and E. faecalis, should be identified as E. durans and E. faecalis by the 16S rDNA analyses, respectively. These 2 strains had equal antimicrobial profile for their type strain. The MIC values of TN-3 and TN-9 for vancomycin were 0.5μg/mL and 1.0μg/mL, and the vancomycin resistance genes were not detected in these 2 strains.