Masakazu Uramoto

Reveromycin A biosynthesis uses RevG and RevJ for stereospecific spiroacetal formation

Spiroacetal compounds are ubiquitous in nature, and their stereospecific structures are responsible for diverse pharmaceutical activities. Elucidation of the biosynthetic mechanisms that are involved in spiroacetal formation will open the door to efficient generation of stereospecific structures that are otherwise hard to synthesize chemically. However, the biosynthesis of these compounds is poorly understood, owing to difficulties in identifying the responsible enzymes and analyzing unstable intermediates. Here we comprehensively describe the spiroacetal formation involved in the biosynthesis of reveromycin A, which inhibits bone resorption and bone metastases of tumor cells by inducing apoptosis in osteoclasts. We performed gene disruption, systematic metabolite analysis, feeding of labeled precursors and conversion studies with recombinant enzymes. We identified two key enzymes, dihydroxy ketone synthase and spiroacetal synthase, and showed in vitro reconstruction of the stereospecific spiroacetal structure from a stable acyclic precursor. Our findings provide insights into the creation of a variety of biologically active spiroacetal compounds for drug leads.

Cytotoxic benzyl dihydroflavonols from Cudrania tricuspidata

Three new dihydroflavonols, gericudranins A-C were isolated from the stem bark of Cudrania tricuspidata. They were identified as 6,8-di-p-hydroxybenzyltaxifolin, 8-p-hydroxybenzyltaxifolin and 6-p-hydroxybenzyltaxifolin, respectively, by means of spectral studies. These compounds were cytotoxic to human tumor cell lines, such as CRL 1579 (skin), LOX-IMVI (skin), MOLT-4F (leukemia), KM12 (colon) and UO-31 (renal) in culture, with ED50 values of 2.7-31.3 micrograms ml-1.

Immunosuppressive activity of a monoterpene from Eucommia ulmoides

Abstract Loliolide has been isolated from the chloroform extract of the leaves of Eucommia ulmoides and shown to have immunosuppressive activity.

Biochemical Characterization of a Novel Indole Prenyltransferase from Streptomyces sp. SN-593

Genome sequencing of Streptomyces species has highlighted numerous potential genes of secondary metabolite biosynthesis. The mining of cryptic genes is important for exploring chemical diversity. Here we report the metabolite-guided genome mining and functional characterization of a cryptic gene by biochemical studies. Based on systematic purification of metabolites from Streptomyces sp. SN-593, we isolated a novel compound, 6-dimethylallylindole (DMAI)-3-carbaldehyde. Although many 6-DMAI compounds have been isolated from a variety of organisms, an enzyme catalyzing the transfer of a dimethylallyl group to the C-6 indole ring has not been reported so far. A homology search using known prenyltransferase sequences against the draft sequence of the Streptomyces sp. SN-593 genome revealed the iptA gene. The IptA protein showed 27% amino acid identity to cyanobacterial LtxC, which catalyzes the transfer of a geranyl group to (-)-indolactam V. A BLAST search against IptA revealed much-more-similar homologs at the amino acid level than LtxC, namely, SAML0654 (60%) from Streptomyces ambofaciens ATCC 23877 and SCO7467 (58%) from S. coelicolor A3(2). Phylogenetic analysis showed that IptA was distinct from bacterial aromatic prenyltransferases and fungal indole prenyltransferases. Detailed kinetic analyses of IptA showed the highest catalytic efficiency (6.13 min(-1) microM(-1)) for L-Trp in the presence of dimethylallyl pyrophosphate (DMAPP), suggesting that the enzyme is a 6-dimethylallyl-L-Trp synthase (6-DMATS). Substrate specificity analyses of IptA revealed promiscuity for indole derivatives, and its reaction products were identified as novel 6-DMAI compounds. Moreover, DeltaiptA mutants abolished the production of 6-DMAI-3-carbaldehyde as well as 6-dimethylallyl-L-Trp, suggesting that the iptA gene is involved in the production of 6-DMAI-3-carbaldehyde.

Identification of Cytochrome P450s Required for Fumitremorgin Biosynthesis in Aspergillus fumigatus

Missing links in the fumitremorgin biosynthetic pathway have been elucidated as 1) hydroxylation of the tryprostatin B indole ring at C‐6 by FtmC, 2) CN bond formation by FtmE to produce fumitremorgin C, and 3) the subsequent dihydroxylation of fumitremorgin C by FtmG to afford fumitremorgin B. These are also crucial processes for the inhibitory activity of fumitremorgin C against breast cancer resistance protein (BCRP).

Chemistry of the neopolyoxins, pyrimidine and imidazoline nucleoside peptide antibiotics

Abstract New chitin synthetase inhibitors, neopolyoxins A, B, and C were isolated from the culture filtrate of Streptomyces cacaoi subsp. asoensis. Their absolute structures have been established on the basis of chemical and spectroscopic evidence. They are structural analogs of the polyoxins. As a nucleobase, neopolyoxin C possesses uracil, while neopolyoxins A and B contain the imidazoline moiety. A ring contraction reaction of pyrimidine nucleoside into imidazoline nucleoside played a key role in the structure determination of the nucleoside moiety. A similar transformation was suggested for the biosynthesis of neopolyoxins A and B.

Structures of neopolyoxins A, B, and C

Abstract The absolute structures of fungal chitin synthetase inhibitors, neopolyoxins A, B, and C were determined as 1, 2, and 3 respectively on the basis of chemical and spectroscopic evidence.

Kerriamycin B inhibits protein SUMOylation

The Journal of Antibiotics (2009) 62, 221–224; doi:10.1038/ja.2009.10; published online 6 March 2009Keywords: kerriamycin B; SUMO-1; SUMO-activating enzyme (E1)Post-translational conjugation of small ubiquitin-related modifierprotein (SUMO) to protein substrates (SUMOylation) hasbeen revealed as one of the major post-translational regulatorysystems in animals and other eukaryotes. SUMO conjugation iscatalyzed by a multi-step enzymatic reaction cascade similar toubiquitinylation.

N-Cyanomethyl-2-chloroisonicotinamide Induces Systemic Acquired Resistance in Arabidopsis without Salicylic Acid Accumulation

Systemic acquired resistance (SAR) is a potent innate immunity system in plants that is induced through the salicylic acid-mediated pathway. N-cyanomethyl-2-chloroisonicotinamide (NCI) is able to induce a broad range of disease resistance in tobacco and rice and induces SAR marker gene expression without SA accumulation in tobacco. To clarify the detailed mode of action of NCI, we analyzed its ability to induce defense gene expression and resistance in Arabidopsis mutants that are defective in various defense signaling pathways. Wild-type Arabidopsis treated with NCI exhibited increased expression of several pathogenesis-related genes and enhanced resistance to the bacterial pathogen, Pseudomonas syringae pv. tomato DC3000. NCI induced disease resistance and PR gene expression in NahG transgenic plants, but not in the npr1 mutant. NCI could induce PR gene expression in the etr1-1, ein2-1 and jar1-1 mutants. Thus, NCI activates SAR, independently from ethylene and jasmonic acid, by stimulating the site between SA and NPR1.

Biosynthesis of the 17-membered carbocyclic ring of lankacidin antibiotics

A mechanism involving contraction of an 18-membered polyketide ring was proposed for the biosynthesis of the 17-membered carbocyclic ring of lankacidin antibiotics based on the feeding experiments of deuterated glycine and 2H-NMR spectroscopy.