Background: Type III polyketide synthases (PKSs) show diverse cyclization specificity. Results: A single amino acid substitution in two Azotobacter type III PKSs reversed their cyclization specificity. Crystal structures were determined. Conclusion: The volume of the active site cavity is a crucial determinant of the cyclization specificity. Significance: An important insight into the cyclization specificity of type III PKSs was provided.

JBIR-76 and -77 are isofuranonaphthoquinones (IFNQs) isolated from Streptomyces sp. RI-77. Draft genome sequencing and gene disruption analysis of Streptomyces sp. RI-77 showed that a type II polyketide synthase (PKS) gene cluster (ifn cluster) was responsible for the biosynthesis of JBIR-76 and -77. It was envisaged that an octaketide intermediate (C16 ) could be synthesized by the minimal PKS (IfnANO) and that formation of the IFNQ scaffold (C13 ) would therefore require a C-C bond cleavage reaction. An ifnQ disruptant accumulated some shunt products (C15 ), which were presumably produced by spontaneous cyclization of the decarboxylated octaketide intermediate. Recombinant IfnQ catalyzed the Baeyer-Villiger oxidation of 1-(2-naphthyl)acetone, an analogue of the bicyclic octaketide intermediate. Based on these results, we propose a pathway for the biosynthesis of JBIR-76 and -77, involving IfnQ-catalyzed C-C bond cleavage as a key step in the formation of the IFNQ scaffold.

Many prenylated indole derivatives are widely distributed in nature. Recently, two Streptomyces prenyltransferases, IptA and its homolog SCO7467, were identified in the biosynthetic pathways for 6-dimethylallylindole (DMAI)-3-carbaldehyde and 5-DMAI-3-acetonitrile, respectively. Here, we isolated a novel prenylated indole derivative, 3-hydroxy-6-dimethylallylindolin (DMAIN)-2-one, based on systematic purification of metabolites from a rare actinomycete, Actinoplanes missouriensis NBRC 102363. The structure of 3-hydroxy-6-DMAIN-2-one was determined by HR-MS and NMR analyses. We found that A. missouriensis produced not only 3-hydroxy-6-DMAIN-2-one but also 6-dimethylallyltryptophan (DMAT) and 6-DMAI when grown in PYM (peptone-yeast extract-MgSO4) medium. We searched the complete genome of A. missouriensis for biosynthesis genes of these compounds and found a gene cluster composed of an iptA homolog (AMIS_22580, named iptA-Am) and a putative tryptophanase gene (AMIS_22590, named tnaA-Am). We constructed a tnaA-Am-deleted (ΔtnaA-Am) strain and found that it produced 6-DMAT but did not produce 6-DMAI or 3-hydroxy-6-DMAIN-2-one. Exogenous addition of 6-DMAI to mutant ΔtnaA-Am resulted in the production of 3-hydroxy-6-DMAIN-2-one. Furthermore, in vitro enzyme assays using recombinant proteins produced by Escherichia coli demonstrated that 6-DMAI was synthesized from tryptophan and dimethylallyl pyrophosphate in the presence of both IptA-Am and TnaA-Am, and that IptA-Am preferred tryptophan to indole as the substrate. From these results, we concluded that the iptA-Am-tnaA-Am gene cluster is responsible for the biosynthesis of 3-hydroxy-6-DMAIN-2-one. Presumably, tryptophan is converted into 6-DMAT by IptA-Am and 6-DMAT is then converted into 6-DMAI by TnaA-Am. 6-DMAI appears to be converted into 3-hydroxy-6-DMAIN-2-one by the function of some unknown oxidases in A. missouriensis.

A novel naphthoquinone-like polyketide, JBIR-85 (1), with a unique skeleton and antioxidative activity was isolated from a culture of Streptomyces sp. RI-77. The planar structure of 1 was established on the basis of extensive NMR and MS analyses. The structure of 1 including the absolute configuration was established via X-ray crystallographic analysis. Since 1 exhibits a unique skeleton, we performed feeding experiments to reconfirm the structure and predict the biosynthetic pathway.