Tokutaro Yamaguchi

Enzymatic synthesis of novel phloretin glucosides.

ABSTRACT A UDP-glycosyltransferase from Bacillus licheniformis was exploited for the glycosylation of phloretin. The in vitro glycosylation reaction confirmed the production of five phloretin glucosides, including three novel glucosides. Consequently, we demonstrated the application of the same glycosyltransferase for the efficient whole-cell biocatalysis of phloretin in engineered Escherichia coli.

Enzymatic synthesis of vancomycin derivatives using galactosyltransferase and sialyltransferase

Analogs of vancomycin and pseudo-vancomycin with new sugar attachments in mono- and di-saccharide form have been enzymatically synthesized by glycosylation with overexpressed glycosyltransferases, β1,4-galactosyltransferase and α2,3-sialyl transferases. All four analogs, including galactose-containing derivatives (6 and 8) and galactose- and sialic acid-containing derivatives (7 and 9) were prepared and characterized by HPLC, LC-MS, NMR and MIC test.

Characterization and structure identification of an antimicrobial peptide, hominicin, produced by Staphylococcus hominis MBBL 2-9

Hominicin, antimicrobial peptide displaying potent activity against Staphylococcus aureus ATCC 25923, methicillin-resistant S. aureus (MRSA) ATCC 11435 and vancomycin-intermediate S. aureus (VISA) CCARM 3501, was purified by chloroform extraction, ion-exchange column chromatography and reverse-phase HPLC from culture supernatant of Staphylococcushominis MBBL 2-9. Hominicin exhibited heat stability up to 121 degrees C for 15min and activity under both acidic and basic conditions (from pH 2.0 to 10.0). Hominicin was cleaved into two fragments after treatment with proteinase K, resulting in the loss of its antibacterial activity, while it was resistant to trypsin, alpha-chymotrypsin, pepsin and lipase. The molecular mass of hominicin determined by mass spectrometry was 2038.4Da. LC-mass spectrometry and NMR spectroscopy analyses of the two fragments revealed the sequence of hominicin as DmIle-Dhb-Pro-Ala-Dhb-Pro-Phe-Dhb-Pro-Ala-Ile-Thr-Glu-Ile-Dhb-Ala-Ala-Val-Ile-Ala-Dmp, which had no similarity with other antimicrobial peptides previously reported. The present study is the first report of this novel antimicrobial peptide, which has uncommon amino acid residues like the ones in Class I group and shows potent activity against clinically relevant S. aureus, MRSA and VISA.

Both extracellular chitinase and a new cyclic lipopeptide, chromobactomycin, contribute to the biocontrol activity of Chromobacterium sp. C61.

Chromobacterium sp. strain C61 displays antifungal activities in vitro and has been used successfully for the biocontrol of plant diseases under field conditions. In this study, the roles of extracellular chitinase and an antifungal compound produced by strain C61 were investigated to elucidate their contributions to biological control activity. The bacterium possessed a locus chi54 encoding an extracellular chitinase, and mutation of chi54 eliminated chitinase production. Production of the extracellular enzyme and expression of the chi54 transcript were increased in the wild-type strain when chitin was added to the culture medium. In vitro assays showed that purified chitinase inhibited spore germination of multiple pathogens. However, the in planta biocontrol activity of filtrates of cultures grown in the presence of chitin was lower than that of filtrates grown without chitin, indicating that correlation between chitinase and biocontrol activity was lacking. The analysis of C61 culture filtrates revealed an antifungal cyclic lipopeptide, chromobactomycin, whose structure contained a unique nonameric peptide ring. The purified chromobactomycin inhibited the growth of several phytopathogenic fungi in vitro, and plant application significantly reduced disease severity for several pathogens. Furthermore, the production of chromobactomycin was reduced in cultures amended with chitin. These data suggest that the production of both the extracellular chitinase Chi54 and the newly identified antibiotic chromobactomycin can contribute, in an interconnected way, to the suppression of plant disease by Chromobacterium sp. strain C61.

Structural modification of herboxidiene by substrate-flexible cytochrome P450 and glycosyltransferase

Herboxidiene is a natural product produced by Streptomyces chromofuscus exhibiting herbicidal activity as well as antitumor properties. Using different substrate-flexible cytochrome P450s and glycosyltransferase, different novel derivatives of herboxidiene were generated with structural modifications by hydroxylation or epoxidation or conjugation with a glucose moiety. Moreover, two isomers of herboxidiene containing extra tetrahydrofuran or tetrahydropyran moiety in addition to the existing tetrahydropyran moiety were characterized. The hydroxylated products for both of these compounds were also isolated and characterized from S. chromofuscus PikC harboring pikC from the pikromycin gene cluster of Streptomyces venezuelae and S. chromofuscus EryF harboring eryF from the erythromycin gene cluster of Saccharopolyspora erythraea. The compounds generated were characterized by high-resolution quadrupole-time-of-flight electrospray ionization mass spectrometry (HR-QTOF-ESI/MS) and 1H- and 13C-nuclear magnetic resonance (NMR) analyses. The evaluation of antibacterial activity against three Gram-positive bacteria, Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus, indicated that modification resulted in a transition from anticancer to antibacterial potency.

A photochemical investigation into operational degradation of arylamines in organic light-emitting diodes

In this study, we investigated the operational degradation of 4,4′-bis(N-carbazolyl)biphenyl (CBP), an arylamine commonly used as phosphorescent host or hole transport material in organic light-emitting diodes (OLEDs), which have been subject to a recent surge in demand for use in the optical display market. In view of the important roles of organic components in the stability and lifetime of OLEDs, we initiated an investigation of the operational degradation of CBP in OLEDs in an effort to elucidate the degradation mechanism. Our experimental approach to this task is by performing photochemistry on CBP in solution, thereby avoiding actual operation of CBP-based OLEDs. Prior to the experiments, we calculated the C–N homolytic bond dissociation energy in CBP, and synthesized two CBP derivatives based on molecular engineering considerations. Furthermore, we performed TiO2 photocatalytic decomposition of arylamines as a feasibility test for another operational degradation pathway. Based on both the photochemical and photocatalytic experiments, multiple operational degradation pathways of arylamines emerge.

Biosynthesis of natural and non-natural genistein glycosides

Genistein is the principal isoflavone constituent of soybean. It has attracted more attention than other plant polyphenols because of its significant affinity with medical interests. Herein, we biosynthesized and structurally characterized ten different natural and non-natural analogues of genistein glycopyranosides using versatile glycosyltransferases (GTs) and sugar-O-methyltransferases (SOMTs). Two GTs, AtUGT89C1 from the Arabidopsis thaliana plant and YjiC from Bacillus licheniformis DSM-13 bacteria, catalyzed the glycosylation reaction. Two SOMTs transferred the methyl group to two specific hydroxy positions in the sugar moiety of genistein rhamnopyranoside, thus diversifying the sugar attached to genistein. Among the biosynthesized compounds, four of the 2-deoxy-D-glycopyranosides, 7-O-(2-deoxy-β-D-arabino-hexopyranosyl)genistein, 4′-O-(2-deoxy-β-D-arabino-hexopyranosyl)genistein, 4′,7-bis-O-(2-deoxy-β-D-arabino-hexopyranosyl)genistein, and 4′,5,7-tri-O-(2-deoxy-β-D-arabino-hexopyranosyl)genistein, and two of the O-methyl rhamnopyranosides, 7-O-(3-O-methyl-α-L-rhamnopyranosyl)genistein and 7-O-(4-O-methyl-α-L-rhamnopyranosyl)genistein, are novel compounds that have not been previously reported. The other glycopyranosides are 7-O-(α-L-rhamnopyranosyl)genistein, 7-O-(β-D-glucopyranosyl)genistein, 4′-O-(β-D-glucopyranosyl)genistein, and 4′,7-O-(β-D-glucopyranosyl)genistein. Microbial production of these novel compounds and other glucopyranosides is appreciable in shake flask culture. This study highlights the application of versatile enzymes in the production of diverse glycosides of medicinally important genistein, which can have positive impacts on a variety of molecular targets in future studies, as shown by genistein with remedied drawbacks.

In vivo characterization of NcsB3 to establish the complete biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore

Neocarzinostatin (NCS) is an enediyne antibiotic produced by Streptomyces carzinostaticus. The NCS chromophore consists of an enediyne core, a sugar moiety, and a naphthoic acid (NA) moiety. The latter plays a key role in binding the NCS chromophore to its apoprotein to protect and stabilize the bioactive NCS chromophore. In this study, we expressed three genes: ncsB (naphthoic acid synthase), ncsB3 (P450 hydroxylase), and ncsB1 (O-methyltransferase), in Streptomyces lividans TK24. The three genes were sufficient to produce 2-hydroxy-7-methoxy-5-methyl-1-naphthoic acid. Production was analyzed and confirmed by LC-MS and nuclear magnetic resonance. Here, we report the functional characterization of ncsB3 and thereby elucidate the complete biosynthetic pathway of NA moiety of the NCS chromophore.

Characterization of sterol glucosyltransferase from Salinispora tropica CNB-440: Potential enzyme for the biosynthesis of sitosteryl glucoside

A sterol glucosyltransferase-encoded gene was isolated from Salinispora tropica CNB-440, a marine, sediment-dwelling, Gram positive bacterium that produces the potent anticancer compound, salinosporamide A. The full-length gene consists of 1284 nucleotides and encodes 427 amino acids with a calculated mass of 45.65kDa. The gene was then cloned and heterologously expressed in Escherichia coli BL21(DE3). The amino acid sequence shares 39% similarity with the glycosyltransferase from Withania somnifera, which belongs to glycosyltransferase family 1. Enzyme reactions were carried out with the various free sterols (acceptor) and NDP-sugars (donor). The purified protein only showed activity for glucosylation of β-sitosterol with UDP-D-glucose and TDP-D-glucose donors, and optimal activity at pH 7.5 and 37°C. Among these two donors, UDP-D-glucose was preferred.

Structure Determination of Macrolactin Compounds with Antibacterial Activities Isolated from Bacillus polyfermenticus KJS-2

In this study, we isolated five macrolactin compounds from a fermentation broth of Bacillus polyfermenticus KJS-2. The macrolactin compounds were structurally identified as macrolactin A (MA), 7-O-malonyl macrolactin A (MMA), 7-O-succinyl macrolactin A (SMA), macrolactin E (ME) and macrolactin F (MF) via a variety of NMR techniques, COZY, DEPT, HMQC and HMBC, and mass and specific rotation assays. The three macrolactin compounds, MA, MMA and SMA, profoundly inhibited the growth of both vancomycin-resistant Enterococci (VREs) and methicillin-resistant Staphylococcus aureus (MRSA), the inhibition of which were estimated via a disc agar diffusion bioassay. MA, MMA, and SMA exhibited antibacterial activities superior to those of vancomycin and teicoplanin.