ABSTRACT
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 typeâ 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.
Subject(s)
Bacterial Proteins/metabolism , Mixed Function Oxygenases/metabolism , Naphthoquinones/metabolism , Streptomyces/chemistry , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Mass Spectrometry , Multigene Family , Naphthoquinones/chemistry , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Streptomyces/metabolismABSTRACT
Type III polyketide synthases (PKSs) show diverse cyclization specificity. We previously characterized two Azotobacter type III PKSs (ArsB and ArsC) with different cyclization specificity. ArsB and ArsC, which share a high sequence identity (71%), produce alkylresorcinols and alkylpyrones through aldol condensation and lactonization of the same polyketomethylene intermediate, respectively. Here we identified a key amino acid residue for the cyclization specificity of each enzyme by site-directed mutagenesis. Trp-281 of ArsB corresponded to Gly-284 of ArsC in the amino acid sequence alignment. The ArsB W281G mutant synthesized alkylpyrone but not alkylresorcinol. In contrast, the ArsC G284W mutant synthesized alkylresorcinol with a small amount of alkylpyrone. These results indicate that this amino acid residue (Trp-281 of ArsB or Gly-284 of ArsC) should occupy a critical position for the cyclization specificity of each enzyme. We then determined crystal structures of the wild-type and G284W ArsC proteins at resolutions of 1.76 and 1.99 Å, respectively. Comparison of these two ArsC structures indicates that the G284W substitution brings a steric wall to the active site cavity, resulting in a significant reduction of the cavity volume. We postulate that the polyketomethylene intermediate can be folded to a suitable form for aldol condensation only in such a relatively narrow cavity of ArsC G284W (and presumably ArsB). This is the first report on the alteration of cyclization specificity from lactonization to aldol condensation for a type III PKS. The ArsC G284W structure is significant as it is the first reported structure of a microbial resorcinol synthase.
Subject(s)
Amino Acid Substitution , Azotobacter vinelandii/enzymology , Bacterial Proteins/chemistry , Polyketide Synthases/chemistry , Polyketides/chemical synthesis , Azotobacter vinelandii/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Catalytic Domain , Mutagenesis, Site-Directed , Polyketide Synthases/genetics , Polyketide Synthases/metabolism , Polyketides/metabolism , Structure-Activity Relationship , Substrate SpecificityABSTRACT
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.
Subject(s)
Free Radical Scavengers/isolation & purification , Naphthoquinones/isolation & purification , Polyketides/isolation & purification , Streptomyces/chemistry , Biphenyl Compounds/pharmacology , Crystallography, X-Ray , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Molecular Conformation , Molecular Structure , Naphthoquinones/chemistry , Nuclear Magnetic Resonance, Biomolecular , Picrates/pharmacology , Polyketides/chemistryABSTRACT
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.