Controlling the production of phytotoxin pyriculol in Pyricularia oryzae by aldehyde reductase.

Pyricularia oryzae is one of the most devastating plant pathogens in the world. This fungus produces several secondary metabolites including the phytotoxin pyriculols, which are classified into 2 types: aldehyde form (pyriculol and pyriculariol) and alcohol form (dihydropyriculol and dihydropyriculariol). Although interconversion between the aldehyde form and alcohol form has been predicted, and the PYC10 gene for the oxidation of alcohol form to aldehyde is known, the gene responsible for the reduction of aldehyde to alcohol form is unknown. Furthermore, previous studies have predicted that alcohol analogs are biosynthesized via aldehyde analogs. Herein, we demonstrated that an aldo/keto reductase PYC7 is responsible for the reduction of aldehyde to alcohol congeners. The results indicate that aldehyde analogs are biosynthesized via alcohol analogs, contradicting the previous prediction. The results suggest that P. oryzae controls the amount of pyriculol analogs using two oxidoreductases, PYC7 and PYC10, thereby controlling the bioactivity of the phytotoxin.

Biosynthetic gene cluster identification and biological activity of lucilactaene from Fusarium sp. RK97-94.

We identified the biosynthetic gene cluster for lucilactaene, a cell cycle inhibitor from a filamentous fungus Fusarium sp. RK 97-94. The luc1 knockout strain accumulated demethylated analogs, indicating the involvement of Luc1 methyltransferase in lucilactaene biosynthesis. Lucilactaene showed potent antimalarial activity. Our data suggested that methylation and ether ring formation are essential for its potent antimalarial activity.

Safety of radiotherapy for hemodialysis patients with cancer.

BACKGROUND: The number of hemodialysis (HD) patients is increasing worldwide, and they are at a higher risk of cancer than the general population. Because HD patients are more likely to have inflammation, radiotherapy (RT)-induced adverse effects (AEs) are theoretically expected to be worse for HD patients. Since only a few reports have been published on this subject, we aimed to evaluate the tolerability of RT in HD patients. METHODS: We retrospectively analyzed AEs related to RT for HD patients. Our study included patients from three institutions treated between January 2007 and July 2017. The patient eligibility criteria were (i) receipt of maintenance HD 2-3 times per week for end-stage renal disease prior to the start of RT and (ii) pathologically confirmed malignancies. The endpoints were acute and late non-hematologic AEs. RESULTS: The study included 56 patients. The most common histology was head and neck cancer (23%), followed by lung cancer (14%) and prostate cancer (11%). The median radiation dose was 60 (range, 12-93.8) Gy at an equivalent dose in 2-Gy fractions (EQD2). The RT completion rate was 96%. Patients had a median follow-up period after RT of 9.1 (range 0.5-98.1) months. Grade 3 or worse acute and late AEs were noted in 6 (11%) and 3 (7%) patients, respectively. In the acute phase, 2 patients had grade 5 AEs, both of which were infections. CONCLUSION: Our results suggest that RT for HD patients is clinically tolerable. However, some patients can experience severe infections related to treatment.

Induction of Nectriapyrone Biosynthesis in the Rice Blast Fungus Pyricularia oryzae by Disturbance of the Two-Component Signal Transduction System.

Most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Nectriapyrones are known as secondary metabolites produced mainly by symbiotic fungi, including endophytes and plant pathogens. Herein, we show the induction of nectriapyrone production in the rice blast fungus Pyricularia oryzae. The two-component signal transduction system was disturbed by disrupting OSM1 and PoYPD1, which encoded a HOG MAP kinase and a His-containing phosphotransfer (HPt) protein, respectively. This induced the production of two polyketide compounds: nectriapyrone and its hydroxylated analogue. The nectriapyrone biosynthetic gene cluster consists of a polyketide synthase gene (NEC1) and an O-methyltransferase gene (NEC2). Overexpression of the two genes induced overproduction of nectriapyrone and five nectriapyrone analogues, including a new derivative. Nectriapyrone production was not required for the infection of rice. The structure of nectriapyrone is similar to that of the germicidins produced by Streptomyces spp., and nectriapyrone inhibited the growth of Streptomyces griseus.

Steroid Degradation in Comamonas testosteroni TA441: Identification of the Entire ß-Oxidation Cycle of the Cleaved B Ring.

Comamonas testosteroni TA441 degrades steroids via aromatization of the A ring, followed by degradation of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid, mainly by ß-oxidation. In this study, we revealed that 7ß,9α-dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-coenzyme A (CoA) ester is dehydrogenated by (3S)-3-hydroxylacyl CoA-dehydrogenase, encoded by scdE (ORF27), and then the resultant 9α-hydroxy-7,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is converted by 3-ketoacyl-CoA transferase, encoded by scdF (ORF23). With these results, the whole cycle of ß-oxidation on the side chain at C-8 of 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid is clarified; 9-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid-CoA ester is dehydrogenated at C-6 by ScdC1C2, followed by hydration by ScdD. 7ß,9α-Dihydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostanoic acid-CoA ester then is dehydrogenated by ScdE to be converted to 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid-CoA ester and acetyl-CoA by ScdF. ScdF is an ortholog of FadA6 in Mycobacterium tuberculosis H37Rv, which was reported as a 3-ketoacyl-CoA transferase involved in C ring cleavage. We also obtained results suggesting that ScdF is also involved in C ring cleavage, but further investigation is required for confirmation. ORF25 and ORF26, located between scdF and scdE, encode enzymes belonging to the amidase superfamily. Disrupting either ORF25 or ORF26 did not affect steroid degradation. Among the bacteria having gene clusters similar to those of tesB to tesR, some have both ORF25- and ORF26-like proteins or only an ORF26-like protein, but others do not have either ORF25- or ORF26-like proteins. ORF25 and ORF26 are not crucial for steroid degradation, yet they might provide clues to elucidate the evolution of bacterial steroid degradation clusters.IMPORTANCE Studies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain materials for steroid drugs. Steroid-degrading bacteria are globally distributed, and the role of bacterial steroid degradation in the environment as well as in relation to human health is attracting attention. The overall aerobic degradation of the four basic steroidal rings has been proposed; however, there is still much to be revealed to understand the complete degradation pathway. This study aims to uncover the whole steroid degradation process in Comamonas testosteroni TA441 as a model of steroid-degrading bacteria. C. testosteroni is one of the most studied representative steroid-degrading bacteria and is suitable for exploring the degradation pathway, because the involvement of degradation-related genes can be determined by gene disruption. Here, we elucidated the entire ß-oxidation cycle of the cleaved B ring. This cycle is essential for the following C and D ring cleavage.

Steroid Degradation in Comamonas testosteroni TA441: Identification of Metabolites and the Genes Involved in the Reactions Necessary before D-Ring Cleavage.

Bacterial steroid degradation has been studied mainly with Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni as representative steroid degradation bacteria for more than 50 years. The primary purpose was to obtain materials for steroid drugs, but recent studies showed that many genera of bacteria (Mycobacterium, Rhodococcus, Pseudomonas, etc.) degrade steroids and that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment. The role of bacterial steroid degradation in the environment is, however, yet to be revealed. To uncover the whole steroid degradation process in a representative steroid-degrading bacterium, C. testosteroni, to provide basic information for further studies on the role of bacterial steroid degradation, we elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage in C. testosteroni TA441. In bacterial oxidative steroid degradation, A- and B-rings of steroids are cleaved to produce 2-hydroxyhexa-2,4-dienoic acid and 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. The latter compound was revealed to be degraded to the coenzyme A (CoA) ester of 9α-hydroxy-17-oxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid, which is converted to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrostan-7-oic acid by ORF31-encoded hydroxylacyl dehydrogenase (ScdG), followed by conversion to the CoA ester of 9,17-dioxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid by ORF4-encoded acyl-CoA dehydrogenase (ScdK). Then, a water molecule is added by the ORF5-encoded enoyl-CoA hydratase (ScdY), which leads to the cleavage of the D-ring. The conversion by ScdG is presumed to be a reversible reaction. The elucidated pathway in C. testosteroni TA441 is different from the corresponding pathways in Mycobacterium tuberculosis H37Rv.IMPORTANCE Studies on representative steroid degradation bacteria Rhodococcus equi (Nocardia restrictus) and Comamonas testosteroni were initiated more than 50 years ago primarily to obtain materials for steroid drugs. A recent study showed that steroid-degrading bacteria are globally distributed and found particularly in wastewater treatment plants, the soil, plant rhizospheres, and the marine environment, but the role of bacterial steroid degradation in the environment is yet to be revealed. This study aimed to uncover the whole steroid degradation process in C. testosteroni TA441, in which major enzymes for steroidal A- and B-ring cleavage were elucidated, to provide basic information for further studies on bacterial steroid degradation. C. testosteroni is suitable for exploring the degradation pathway because the involvement of degradation-related genes can be determined by gene disruption. We elucidated the two indispensable oxidative reactions and hydration before D-ring cleavage, which appeared to differ from those present in Mycobacterium tuberculosis H37Rv.

Total Synthesis of Highly Oxygenated Bisabolane Sesquiterpene Isolated from Ligularia lankongensis: Relative and Absolute Configurations of the Natural Product.

The relative and absolute configurations of an oxygenated bisabolane natural product, isolated from Ligularia lankongensis, were determined by synthesis. All four possible stereoisomers and their tiglate analogues were synthesized from R-(-)-carvone, and their 1H and 13C NMR spectra were compared to establish the 6R,8S,10S configuration. The stereoselective synthesis of the natural product was also achieved, featuring Brown allylation, vanadium-catalyzed epoxidation, and the Mitsunobu reaction.

Development of SAAP3D force field and the application to replica-exchange Monte Carlo simulation for chignolin and C-peptide.

Single amino acid potential (SAAP) would be a prominent factor to determine peptide conformations. To prove this hypothesis, we previously developed SAAP force field for molecular simulation of polypeptides. In this study, the force field was renovated to SAAP3D force field by applying more accurate three-dimensional main-chain parameters, instead of the original two-dimensional ones, for the amino acids having a long side-chain. To demonstrate effectiveness of the SAAP3D force field, replica-exchange Monte Carlo (REMC) simulation was performed for two benchmark short peptides, chignolin (H-GYDPETGTWG-OH) and C-peptide (CHO-AETAAAKFLRAHA-NH2). For chignolin, REMC/SAAP3D simulation correctly produced native ß-turn structures, whose minimal all-atom root-mean-square deviation value measured from the native NMR structure (except for H) was 1.2 Å, at 300 K in implicit water, along with misfolded ß-hairpin structures with unpacked aromatic side chains of Tyr2 and Trp9. Similar results were obtained for chignolin analog [G1Y,G10Y], which folded more tightly to the native ß-turn structure than chignolin did. For C-peptide, on the other hand, the α-helix content was larger than the ß content on average, suggesting a significant helix-forming propensity. When the imidazole side chain of His12 was protonated (i.e., [His12Hip]), the α content became larger. These observations as well as the representative structures obtained by clustering analysis were in reasonable agreement not only with the structures of C-peptide that were determined in this study by NMR in 30% CD3CD in H2O at 298 K but also with the experimental and theoretical behaviors having been reported for protonated C-peptide. Thus, accuracy of the SAAP force field was improved by applying three-dimensional main-chain parameters, supporting prominent importance of SAAP for peptide conformations.

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.

Haenamindole, an unusual diketopiperazine derivative from a marine-derived Penicillium sp. KCB12F005.

During the chemical investigation of marine-derived fungus, an unusual diketopiperazine (DKP) alkaloid, haenamindole (1), was isolated from a culture of the marine-derived fungus Penicillium sp. KCB12F005. The structure of 1, which possesses benzyl-hydroxypiperazindione and phenyl-pyrimidoindole rings system in the molecule, was elucidated by analysis of NMR and MS data. The stereochemistry of 1 was determined by ROESY and advanced Marfey's method.

Identification of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid in steroid degradation by Comamonas testosteroni TA441 and its conversion to the corresponding 6-en-5-oyl coenzyme A (CoA) involving open reading frame 28 (ORF28)- and ORF30-encoded acyl-CoA dehydrogenases.

Comamonas testosteroni TA441 degrades steroids via aromatization and meta-cleavage of the A ring, followed by hydrolysis, and produces 9,17-dioxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid as an intermediate compound. Herein, we identify a new intermediate compound, 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid. Open reading frame 28 (ORF28)- and ORF30-encoded acyl coenzyme A (acyl-CoA) dehydrogenase was shown to convert the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrostan-5-oic acid to the CoA ester of 9α-hydroxy-17-oxo-1,2,3,4,10,19-hexanorandrost-6-en-5-oic acid. A homology search of the deduced amino acid sequences suggested that the ORF30-encoded protein is a member of the acyl-CoA dehydrogenase_fadE6_17_26 family, whereas the deduced amino acid sequence of ORF28 showed no significant similarity to specific acyl-CoA dehydrogenase family proteins. Possible steroid degradation gene clusters similar to the cluster of TA441 appear in bacterial genome analysis data. In these clusters, ORFs similar to ORFs 28 and 30 are often found side by side and ordered in the same manner as ORFs 28 and 30.

Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5-microtubule interaction and in several Eg5 mutants. 11-Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule-stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S-trityl-L-cysteine (STLC) and GSK-1, both terpendole E and 11-ketopaspaline only partially inhibited Eg5-microtubule interaction. Furthermore, terpendole E and 11-ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5(D130A), Eg5(L214A)) or GSK-1 (Eg5(I299F), Eg5(A356T)), but with the same extent of inhibition against wild-type Eg5. Because Eg5(D130A) and Eg5(L214A) show cross-resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop-binding type Eg5 inhibitors.

Application of plug-plug technique to ACE experiments for discovery of peptides binding to a larger target protein: a model study of calmodulin-binding fragments selected from a digested mixture of reduced BSA.

To discover peptide ligands that bind to a target protein with a higher molecular mass, a concise screening methodology has been established, by applying a "plug-plug" technique to ACE experiments. Exploratory experiments using three mixed peptides, mastoparan-X, ß-endorphin, and oxytocin, as candidates for calmodulin-binding ligands, revealed that the technique not only reduces the consumption of the protein sample, but also increases the flexibility of the experimental conditions, by allowing the use of MS detection in the ACE experiments. With the plug-plug technique, the ACE-MS screening methodology successfully selected calmodulin-binding peptides from a random library with diverse constituents, such as protease digests of BSA. Three peptides with Kd values between 8-147 µM for calmodulin were obtained from a Glu-C endoprotease digest of reduced BSA, although the digest showed more than 70 peaks in its ACE-MS electropherogram. The method established here will be quite useful for the screening of peptide ligands, which have only low affinities due to their flexible chain structures but could potentially provide primary information for designing inhibitors against the target protein.

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.

Identification of amino acid residues essential for onion lachrymatory factor synthase activity.

Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H(+) substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23-169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23-169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket.

Possible molecular mechanisms of species recognition by barnacle larvae inferred from multi-specific sequencing analysis of proteinaceous settlement-inducing pheromone.

Gregarious settlement is essential for reproduction and survival of many barnacles. A glycoprotein, settlement-inducing protein complex (SIPC) has been recognized as a signal for settlement and it is expressed in both conspecific adults and larvae. Although the settlement-inducing activities of SIPC are species-specific, the molecular-based mechanism by which larvae distinguish conspecific SIPC from the SIPC of other species is still unknown. Here, the complete primary structure of the SIPC of Megabalanus coccopoma, as well as the partial structure of the SIPCs of Balanus improvisus, Megabalanus rosa, and Elminius modestus are reported. These SIPCs contain highly variable regions that possibly modulate the affinity for the receptor, resulting in the species specificity of SIPC. In addition, the distribution patterns of potential N-glycosylation sites were seen to be different among the various species. Differences in such post-translational modifications may contribute to the species specificity of SIPC.

Chemical and genetic study of Ligularia duciformis and related species in Sichuan and Yunnan Provinces of China.

The chemical constituents of the root extracts and the evolutionarily neutral DNA base sequences were studied for 28 samples of Ligularia duciformis, L. kongkalingensis, and L. nelumbifolia collected in Sichuan and Yunnan Provinces of China. The samples could be classified into four chemotypes (1-4). Sesquiterpenoids having eremophilane and oplopane skeletons were isolated from two (Chemotype 1) and three (Chemotype 2) samples, respectively. Two new oplopane derivatives were isolated and their structures were determined. In 18 samples, phenylpropenoids were the major components (Chemotype 3). In five samples, neither phenylpropenoids nor sesquiterpenoids were found (Chemotype 4). Despite this large chemical variety, no correlation was found between the chemotype and the morphological criteria of species identification. The analysis of the evolutionarily neutral DNA regions also indicated that the samples were not separated into distinct clades and that introgression was extensive.

Zinc suppresses Th17 development via inhibition of STAT3 activation.

Zinc (Zn) is an essential trace metal required by many enzymes and transcription factors for their activity or the maintenance of their structure. Zn has a variety of effects in the immune responses and inflammation, although it has not been well known how Zn affects these reactions on the molecular basis. We here showed that Zn suppresses T(h)17-mediated autoimmune diseases at lest in part by inhibiting the development of T(h)17 cells via attenuating STAT3 activation. In mice injected with type II collagen to induce arthritis, Zn treatment inhibited T(h)17 cell development. IL-6-mediated activation of STAT3 and in vitro T(h)17 cell development were all suppressed by Zn. Importantly, Zn binding changed the alpha-helical secondary structure of STAT3, disrupting the association of STAT3 with JAK2 kinase and with a phospho-peptide that included a STAT3-binding motif from the IL-6 signal transducer gp130. Thus, we conclude that Zn suppresses STAT3 activation, which is a critical step for T(h)17 development.

Isolation of salsolinol, a tetrahydroisoquinoline alkaloid, from the marine sponge Xestospongia cf. vansoesti as a proteasome inhibitor.

Salsolinol (1), a tetrahydroisoquinoline alkaloid, was isolated from the marine sponge Xestospongia cf. vansoesti collected in Indonesia as a proteasome inhibitor, along with three salsolinol derivatives, norsalsolinol (2), cis-4-hydroxysalsolinol (3), and trans-4-hydroxysalsolinol (4). Compounds 1 and 2 inhibited the chymotrypsin-like activity of the proteasome with IC(50) values of 50 and 32 µg/ml, respectively, but 3 and 4 showed no inhibitory effect even at 100 µg/ml.

Solution structure of polytheonamide B, a highly cytotoxic nonribosomal polypeptide from marine sponge.

Polytheonamide B (pTB), a highly cytotoxic polypeptide, is one of the most unusual nonribosomal peptides of sponge origin. pTB is a linear 48-residue peptide with alternating D- and L-amino acids and contains a total of eight types of nonproteinogenic amino acids. To investigate the mechanisms underlying its cytotoxic activity, we determined the three-dimensional structure of pTB by NMR spectroscopy, structure calculation, and energy minimization. pTB adopts a single right-handed ß(6.3)-helical structure in a 1:1 mixture of methanol/chloroform with a length of approximately 45 A and a hydrophilic pore of ca. 4 A inner diameter. These features indicate that pTB molecules form transmembrane channels that permeate monovalent cations as gramicidin A channels do. The strong cytotoxicity of pTB can be ascribed to its ability to form single molecule channels through biological membranes.