Toshihiko Nogawa

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.

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.

Xanthohumol Impairs Autophagosome Maturation through Direct Inhibition of Valosin-Containing Protein

Autophagy is a bulk, nonspecific protein degradation pathway that is involved in the pathogenesis of cancer and neurodegenerative disease. Here, we observed that xanthohumol (XN), a prenylated chalcone present in hops (Humulus lupulus L.) and beer, modulates autophagy. By using XN-immobilized beads, valosin-containing protein (VCP) was identified as a XN-binding protein. VCP has been reported to be an essential protein for autophagosome maturation. Using an in vitro pull down assay, we showed that XN bound directly to the N domain, which is known to mediate cofactor and substrate binding to VCP. These data indicated that XN inhibited the function of VCP, thereby allowing the impairment of autophagosome maturation and resulting in the accumulation of microtubule-associated protein 1 light chain 3-II (LC3-II). This is the first report demonstrating XN as a VCP inhibitor that binds directly to the N domain of VCP. Our finding that XN bound to and inactivated VCP not only reveals the molecular mechanism of XN-modulated autophagy but may also explain how XN exhibits various biological activities that have been reported previously.

Construction of a microbial natural product library for chemical biology studies.

The RIKEN Natural Products Depository (NPDepo) is a public depository of small molecules. Currently, the NPDepo chemical library contains 39,200 pure compounds, half of which are natural products and their derivatives. In order to reinforce the uniqueness of our chemical library, we have improved our strategies for the collection of microbial natural products. Firstly, a microbial metabolite fraction library coupled with an MP (microbial products) plot database provides a powerful resource for the efficient isolation of microbial metabolites. Secondly, biosynthetic studies of microbial metabolites have enabled us to not only access ingenious biosynthetic machineries, but also obtain a variety of biosynthetic intermediates. Our chemical library contributes to the discovery of molecular probes for increasing our understanding of complex biological processes and for eventually developing new drug leads.

A new enzyme involved in the control of the stereochemistry in the decalin formation during equisetin biosynthesis

Tetramic acid containing a decalin ring such as equisetin and phomasetin is one of the characteristic scaffolds found in fungal bioactive secondary metabolites. Polyketide (PKS)-nonribosomal peptide synthetase (NRPS) hybrid enzyme is responsible for the synthesis of the polyketide scaffold conjugated with an amino acid. PKS-NRPS hybrid complex programs to create structural diversity in the polyketide backbone have begun to be investigated, yet mechanism of control of the stereochemistry in a decalin formation via a Diels-Alder cycloaddition remains uncertain. Here, we demonstrate that fsa2, which showed no homology to genes encoding proteins of known function, in the fsa cluster responsible for equisetin and fusarisetin A biosynthesis in Fusarium sp. FN080326, is involved in the control of stereochemistry in decalin formation via a Diels-Alder reaction in the equisetin biosynthetic pathway.

Verticilactam, a New Macrolactam Isolated from a Microbial Metabolite Fraction Library

Systematic isolation of microbial metabolites has been performed to construct microbial metabolite libraries or fraction libraries. A novel macrolactam, verticilactam (1), was isolated from a library of Streptomyces spiroverticillatus JC-8444. The structure was determined on the basis of NMR and mass spectrometric measurements. 1 had a unique 16-membered macrolactam skeleton including a β-keto-amide moiety.

Pyrrolizilactone, a new pyrrolizidinone metabolite produced by a fungus

In the course of our screening program to find structurally unique metabolites from microorganisms on the basis of spectral data collected through LC/MS analysis, a new pyrrolizidinone metabolite, pyrrolizilactone (1) (Figure 1), was discovered and isolated from an uncharacterized fungus. The structure of 1 was determined from spectroscopic results. Compound 1 showed moderate cytotoxic activity against HL-60 and HeLa cells. Microorganisms have a tremendous capacity for producing structurally diverse metabolites, which show various activities.1 They are important sources of pharmaceutical leads and therapeutic agents,2,3 and are also used as bioprobes in chemical biology for the exploration of biological functions.4,5 To search for and discover such structurally unique metabolites efficiently and rapidly, we have constructed a microbial metabolite fraction library with a spectral database on the basis of photodiode array detectorattached LC/MS analysis.6,7 Through our methodology for the construction of this fraction library, we discovered and identified a 16-membered macrolactam with an unusual b-keto-amide moiety, verticilactam,8 6,6-spiroacetal polyketide, spirotoamides, A and B,9 the new fraquinocins, I and J,10 and 6-dimethylallylindole-3carbaldehyde.11 These results demonstrate the advantage of the fraction library in isolating novel metabolites from natural sources. We report herein the isolation of a novel compound from a fungi fraction library. An 18-liter culture broth of an uncharacterized fungus was cultivated to obtain 16.1 g of an ethyl acetate-soluble extract. This was separated into eight fractions through a silica-gel column chromatography, with a stepwise gradient of CHCl3/MeOH. The second fraction eluted with CHCl3/MeOH (100:1) was further separated by chromatography using a Sephadex LH-20 column with CHCl3/MeOH (1:1) to afford three fractions. The second fraction, showing an unidentified peak in LC/MS analysis, was purified by C18HPLC to afford a colorless amorphous solid (1, 5.5 mg). Colorless amorphous; [a]589þ 5.91 (c 0.08, MeOH); UV (MeOH) lmax (log e) 209 (3.34), 236 (3.01) nm; IR (ATR) nmax (cm 1) 3410, 2920, 2875, 1790, 1715, 1685, 1575, 1450, 1375, 1335, 1280, 1160, 1110, 1020; 1H NMR and 13C NMR data, see Table 1; HRESIMS m/z: 416.2433 [MþH]þ (calcd for C24H34NO5: 416.2437). Compound 1 had the molecular formula C24H33NO5, as determined by HRESIMS. The IR spectrum implied the presence of hydroxyl (3410 cm 1) and carbonyl (1685, 1715 and 1790 cm 1) groups. The 1H NMR spectrum showed five methyl signals, which included a singlet, three doublets and a doublet of doublet (1.71 p.p.m., dd, J1⁄4 0.9, 0.9 Hz) branched at an sp2 carbon, and an olefin signal (5.12 p.p.m., br s), suggesting that 1 contained a double bond (Supplementary Figure S1). The 13C NMR spectrum showed 24 signals including five methyls, four methylenes, nine methines (including oxygenated and olefin carbons: 81.6 and 131.3 p.p.m.),

Spirotoamides A and B, novel 6,6-spiroacetal polyketides isolated from a microbial metabolite fraction library

Two new 6,6-spiroacetal polyketides, spirotoamides A (1) and B (2), were isolated from a microbial metabolite fraction library of Streptomyces griseochromogenes JC82-1223 by screening of structurally unique compounds based on a search of spectral database. The fraction library was constructed using a systematic separation method to efficiently discover new metabolites from microbial sources such as actinomycetes and fungi. The structures of 1 and 2 were elucidated by 2D-NMR and mass spectrometric measurements. They belong to a class of polyketides, and contain a 6,6-spiroacetal core structure and a carboxamide group. The biosynthetic pathway of 1 and 2 is discussed in the text.

RK-1355A and B, novel quinomycin derivatives isolated from a microbial metabolites fraction library based on NPPlot screening.

Two novel quinomycin derivatives, RK-1355A (1) and B (2), and one known quinomycin derivative, UK-63,598 (3), were isolated from a microbial metabolites fraction library of Streptomyces sp. RK88-1355 based on Natural Products Plot screening. The structural elucidation of 1 and 2 was established through two-dimensional NMR and mass spectrometric measurements. They belong to a class of quinomycin antibiotics family having 3-hydroxyquinaldic acid and a sulfoxide moiety. They are the first examples for natural products as a quinoline type quinomycin having a sulfoxide on the intramolecular cross-linkage. They showed potent antiproliferative activities against various cancer cell lines and they were also found to exhibit moderate antibacterial activity.

Identification of a molecular target of a novel fungal metabolite, pyrrolizilactone, by phenotypic profiling systems.

In the course of screening our microbial metabolite fraction library, we identified a novel pyrrolizidinone compound, pyrrolizilactone. In this study, we report the identification and characterization of a molecular target for pyrrolizilactone by using two phenotypic profiling systems. Cell morphology‐based profiling analysis using an imaging cytometer (MorphoBase) classified pyrrolizilactone as a proteasome inhibitor. Consistently, proteome‐based profiling analysis using 2D difference gel electrophoresis (DIGE; ChemProteoBase) also demonstrated that pyrrolizilactone is associated with proteasome inhibition. On the basis of these predictions, we determined that pyrrolizilactone is a novel type of proteasome inhibitor inhibiting the trypsin‐like activity of the proteasome.