Characterization and Functional Modification of StaC and RebC, Which Are Involved in the Pyrrole Oxidation of Indolocarbazole Biosynthesis

被引：18

作者：

Asamizu, Shumpei ^{[2
]}

Shiro, Yoshitsugu ^{[1
]}

Igarashi, Yasuhiro ^{[2
,3
]}

Nagano, Shingo ^{[1
,4
]}

Onaka, Hiroyasu ^{[2
,3
]}

机构：

[1] RIKEN SPring 8 Ctr, Biomet Sci Lab, Mikazuki, Hyogo 6795148, Japan

[2] Toyama Prefectural Univ, Dept Biotechnol, Fac Engn, Toyama 9390398, Japan

[3] Toyama Prefectural Univ, Biotechnol Res Ctr, Toyama 9390398, Japan

[4] Tottori Univ, Dept Chem & Biotechnol, Grad Sch Engn, Tottori 6808552, Japan

来源：

BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY | 2011年 / 75卷 / 11期

基金：

日本学术振兴会;

关键词：

indolocarbazole biosynthesis; Streptomyces; secondary metabolism; FAD-dependent monooxygenase; mutational analysis; STREPTOMYCES SP TP-A0274; PROTEIN-KINASE-C; GENE-CLUSTER; CHROMOPYRROLIC ACID; CYTOCHROME-P450; STAP; REBECCAMYCIN; STAUROSPORINE; EXPRESSION; PRODUCTS; COFACTOR;

D O I：

10.1271/bbb.110474

中图分类号：

Q5 [生物化学]; Q7 [分子生物学];

学科分类号：

071010 ; 081704 ;

摘要：

The diversity of indolocarbazole natural products results from the differences in oxidation states of the pyrroline ring moiety. In the biosynthetic pathways for staurosporine and rebeccamycin, two homologous enzymes having 64% identity, StaC and RebC, are responsible for the selective production of K252c, which has one oxo group at the pyrroline ring, and arcyriaflavin A, which has two. Although StaC has a FAD-binding motif, most StaC molecules do not contain FAD, and the protein cannot be reconstituted with FAD in vitro. In this study, we mutated Ala-118 in StaC by replacing a glutamine that is conserved in FAD monooxygenases, resulting in increased FAD content as well as catalytic activity. In addition, mutations around the substrate-binding sites of StaC and RebC can change the product selectivity. Specifically, StaC-N244R-V246T and RebC-F216V-R239N mutants produced substantial amounts of arcyriaflavin A and K252c, respectively.

引用

页码：2184 / 2193

页数：10

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