Masashi Ueki

A Novel Action of Terpendole E on the Motor Activity of Mitotic Kinesin Eg5

To reveal the mechanism of mitosis, the development of M phase-specific inhibitors is an important strategy. We have been screening microbial products to find specific M phase inhibitors that do not directly target tubulins, and rediscovered terpendole E (TerE) as a novel Eg5 inhibitor. TerE did not affect microtubule integrity in interphase, but induced formation of a monoastral spindle in M phase. TerE inhibited both motor and microtubule-stimulated ATPase activities of human Eg5, but did not affect conventional kinesin from either Drosophila or bovine brain. Although terpendoles have been reported as inhibitors of acyl-CoA:cholesterol O-acyltransferase (ACAT), the Eg5 inhibitory activity of TerE was independent of ACAT inhibition. Taken together, we demonstrate that TerE is a novel Eg5 inhibitor isolated from a fungal strain.

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.

4-Isoavenaciolide covalently binds and inhibits VHR, a dual-specificity phosphatase

A potent inhibitor of a dual‐specificity protein phosphatase, VHR (vaccinia H1 related), was isolated during a screening of microbial metabolites. This inhibitor was identified as 4‐isoavenaciolide (4‐iA), and was determined to irreversibly inhibit VHR phosphatase activity with a 50% inhibitory concentration of 1.2 μM. Detailed tandem mass spectrometry analyses of proteolysed fragments revealed that two molecules of 4‐iA bound a molecule of VHR at the two different fragments: one containing the catalytic domain and the other containing the α6 helix positioned surface domain. As 4‐iA possesses a reactive exo‐methylene moiety, it is possible that 4‐iA inhibits VHR through the direct binding to the cysteine residue in the catalytic site (Cys124). Furthermore, 4‐iA inhibited dual‐specificity protein phosphatases and tyrosine phosphatases, but did not inhibit serine/threonine phosphatases. These results suggest that 4‐iA is a cysteine‐targeting inhibitor of protein phosphatases with a common HCX5RS/T motif in the catalytic site.

Terpendole E, a Kinesin Eg5 Inhibitor, Is a Key Biosynthetic Intermediate of Indole-Diterpenes in the Producing Fungus Chaunopycnis alba

Terpendole E is the first natural product inhibitor of kinesin Eg5. Because terpendole E production is unstable, we isolated and analyzed the terpendole E biosynthetic gene cluster, which consists of seven genes encoding three P450 monooxygenases (TerP, TerQ, and TerK), an FAD-dependent monooxygenase (TerM), a terpene cyclase (TerB), and two prenyltransferases (TerC and TerF). Gene knockout and feeding experiments revealed that terpendole E is a key intermediate in terpendole biosynthesis and is produced by the action of the key enzyme TerQ from paspaline, a common biosynthetic intermediate of indole-diterpenes. TerP converts terpendole E to a downstream intermediate specific to terpendole biosynthesis and converts paspaline to shunt metabolites. We successfully overproduced terpendole E by disrupting the terP gene. We propose that terpendole E is a key biosynthetic intermediate of terpendoles and related indole-diterpenes.

Characterization of Giant Modular PKSs Provides Insight into Genetic Mechanism for Structural Diversification of Aminopolyol Polyketides

Polyketides form many clinically valuable compounds. However, manipulation of their biosynthesis remains highly challenging. An understanding of gene cluster evolution provides a rationale for reprogramming of the biosynthetic machinery. Herein, we report characterization of giant modular polyketide synthases (PKSs) responsible for the production of aminopolyol polyketides. Heterologous expression of over 150 kbp polyketide gene clusters successfully afforded their products, whose stereochemistry was established by taking advantage of bioinformatic analysis. Furthermore, phylogenetic analysis of highly homologous but functionally diverse domains from the giant PKSs demonstrated the evolutionary mechanism for structural diversification of polyketides. The gene clusters characterized herein, together with their evolutionary insights, are promising genetic building blocks for de novo production of unnatural polyketides.

Boseongazepines A-C, pyrrolobenzodiazepine derivatives from a Streptomyces sp. 11A057.

Three new pyrrolobenzodiazepine derivatives, boseongazepines A-C (1-3), were isolated from a culture broth of Streptomyces sp. 11A057, together with the known compound usabamycin B (4). The structures of 1-4 were determined through the analysis of spectroscopic data including extensive 1D-, 2D-NMR, and MS techniques. Cell growth inhibition effects of these compounds were evaluated against Jurkat, K-562, HL-60, and HepG2 cell lines.

Structures and biological activities of azaphilones produced by Penicillium sp. KCB11A109 from a ginseng field.

Twelve metabolites, including five highly oxygenated azaphilones, geumsanols A-E, along with seven known analogues were isolated from Penicillium sp. KCB11A109, a fungus derived from a ginseng field. Their structures were assigned by spectroscopic means (NMR and MS), and stereochemistries were determined by extensive spectroscopic analyses ((1)H-(1)H coupling constants, NOESY, and HETLOC) and chemical derivatizations (modified Moshers method and acetonide formation). The isolates were evaluated for their anticancer, antimicrobial, antimalarial activities, and phenotypic effects in zebrafish development. Of these compounds possessing no pyranoquinone core, only geumsanol E exhibited cytotoxic activities and toxic effects on zebrafish embryos, suggesting that a double bond at C-11 and C-12 is important for biological activity.

Deamino-hydroxy-phoslactomycin B, a biosynthetic precursor of phoslactomycin, induces myeloid differentiation in HL-60 cells

During the screening for novel differentiation inducers, we found that a culture broth of Streptomyces sp. HK-803 induced myeloid differentiation of HL-60 cells. The active substance was identified as deamino-hydroxy-phoslactomycin B (HPLM) by mass spectrometry, and synthesized HPLM also induced the differentiation of HL-60 cells. HPLM showed greater inhibition of protein phosphatase 2A (PP2A) activity than phoslactomycin B (PLMB); however, PLMB and okadaic acid did not induce differentiation. Moreover, treatment with ATRA and 1alpha, 25(OH)2D3 induced retinoic acid receptor-beta and 1alpha, 25(OH)2D3 24-hydroxylase, respectively, whereas HPLM did not, suggesting that HPLM is a novel differentiation inducer.

Identification of novel secreted fatty acids that regulate nitrogen catabolite repression in fission yeast

Uptake of poor nitrogen sources such as branched-chain amino acids is repressed in the presence of high-quality nitrogen sources such as NH4+ and glutamate (Glu), which is called nitrogen catabolite repression. Amino acid auxotrophic mutants of the fission yeast Schizosaccharomyces pombe were unable to grow on minimal medium containing NH4Cl or Glu even when adequate amounts of required amino acids were supplied. However, growth of these mutant cells was recovered in the vicinity of colonies of the prototrophic strain, suggesting that the prototrophic cells secrete some substances that can restore uptake of amino acids by an unknown mechanism. We identified the novel fatty acids, 10(R)-acetoxy-8(Z)-octadecenoic acid and 10(R)-hydroxy-8(Z)-octadecenoic acid, as secreted active substances, referred to as Nitrogen Signaling Factors (NSFs). Synthetic NSFs were also able to shift nitrogen source utilization from high-quality to poor nitrogen sources to allow adaptive growth of the fission yeast amino acid auxotrophic mutants in the presence of high-quality nitrogen sources. Finally, we demonstrated that the Agp3 amino acid transporter was involved in the adaptive growth. The data highlight a novel intra-species communication system for adaptation to environmental nutritional conditions in fission yeast.

Isolation of new polyketide metabolites, linearolides A and B, from Streptomyces sp. RK95-74

Although all Streptomyces strains are now thought to have 20–30 gene clusters for secondary metabolite biosynthesis, we cannot actually identify so many kinds of metabolites from one strain by conventional methods. Using Streptomyces sp. RK95-74, previously found as a cytotrienin producer, we searched new metabolites other than cytotrienin derivatives. Following the cultivation with new media and the peak-guided fractionation, we have found new compounds with new polyketide scaffold, named linearolides A and B.