title: Characterization and functional modification of StaC and RebC, which are involved in the pyrrole oxidation of indolocarbazole biosynthesis.
abstract: 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.
authors: Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, Toyama, Japan.
authors: Shumpei
authors: S
authors: Asamizu
authors: Yoshitsugu
authors: Y
authors: Shiro
authors: Yasuhiro
authors: Y
authors: Igarashi
authors: Shingo
authors: S
authors: Nagano
authors: Hiroyasu
authors: H
authors: Onaka
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002227:Carbazoles / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D026121:Indole Alkaloids / Q000737:chemistry / Q000378:metabolism*; D007211:Indoles / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism; D011758:Pyrroles / Q000737:chemistry; D019311:Staurosporine / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D002227:Carbazoles; D026121:Indole Alkaloids; D007211:Indoles; D011758:Pyrroles; C089657:arcyriaflavin A; D005182:Flavin-Adenine Dinucleotide; C053339:rebeccamycin; C049985:staurosporine aglycone; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming); D019311:Staurosporine