Hiroshi Oyama

A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31

A Novel Laccase Inhibitor, N-Hydroxyglycine, Produced by Penicillium citrinum YH-31 Sawao Murao, Yuji Hinode, Eiko Matsumura, Atushi Numata, Kenzo Kawai, Hirofumi Ohishi, Hisanori Jin, Hiroshi Oyama & Takashi Shin a Department of Applied Microbial Technology, The Kumamoto Institute of Technology, Ikeda 4–22–1, Kumamoto 860, Japan b Osaka University of Pharmaceutical Sciences, Matsubara, Osaka 580, Japan Published online: 12 Jun 2014.

Purification and Characterization of Arctium lappa L. (Edible Burdock) Polyphenol Oxidase

Polyphenol oxidase of Arctium lappa L. (edible burdock) has been purified by chromatographies on DEAE-cellulose, Sephadex G-75, and phenyl-Cellulofine to a homogeneous state as judged by SDS polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was estimated to be 31,000 by SDS-PAGE and 25,000 by gel filtration on TSKgel G2000SW. The optimum pH was 7.0 and the enzyme was stable at pH 7.0-9.0. The enzyme oxidized triphenols such as pyrogallol and phloroglucinol, and was completely inhibited by sulfide and cyanide, while it was neither affected by kojic acid nor N-hydroxyglycine (laccase inhibor). These results indicated that the enzyme had properties different from those of polyphenol oxidases from other sources such as mandarin orange and soybean.

Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

Purification and Characterization of an Alkaline Proteinase Produced by Pimelobacter sp. Z-483

An extracellular alkaline proteinase was purified from the culture filtrate of Pimelobacter sp. Z-483. The molecular mass of the purified enzyme was estimated to be approximately 23 kDa by SDS-PAGE and 27 kDa by gel filtration. The optimum pH and temperature for the hydrolysis of casein were approximately 9 and 50°C, respectively. The enzyme activity was strongly inhibited by 1,10-phenanthroline and mercury chloride, but not by EDTA, phosphoramidon and Zincov. The amino terminal sequence of the enzyme was similar to that of an alkaline elastase (MAP1) from Myxococcus xanthus. However, the specificity of the alkaline proteinase in the cleavage of the oxidized insulin B-chain was different from those of the Myxococcus enzyme and animal elastase.

Purification and Characterization of a Prolyl Endopeptidase from Pseudomonas sp. KU-22

Abstract We detected a prolyl endopeptidase (EC 3.4.21.26) in the cell-free extract of Pseudomonas sp. KU-22. The enzyme was purified approximately 2,000-fold by a series of column chromatographies. The molecular weight and isoelectric point of the purified enzyme were estimated to be 76,000 and pH 4.9, respectively. The enzyme was most active at 45°C and pH 8.0 with benzyloxycarbonyl- l -alanyl- l -alanyl- l -proline p -nitroanilide (Z-Ala-Ala-Pro-NphNO 2 ) as a substrate. Significant inhibition of the enzyme by DFP and prolyl endopeptidase-specific inhibitors was observed. Some properties noted, particularly pH stability, amino acid composition, and pI of the enzyme, are clearly different from those of other bacterial prolyl endopeptidases reported to date, indicating that the enzyme is a member of the microbial prolyl endopeptidase family.

Oral Administration of Collagen Hydrolysates Improves Glucose Tolerance in Normal Mice Through GLP-1-Dependent and GLP-1-Independent Mechanisms

The aim of this study was to evaluate the antidiabetic properties of collagen hydrolysates (CHs). CHs exhibited dipeptidyl peptidase-IV inhibitory activity and stimulated glucagon-like-peptide-1 (GLP-1) secretion in vitro. We also determined whether CHs improve glucose tolerance in normal mice. Oral administration of CHs suppressed the glycemic response during the oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT), but the effects were weaker in IPGTT than in OGTT. CHs had no effect on the gastric emptying rate. A pretreatment with the GLP-1 receptor antagonist, exendin 9-39 (Ex9), partially reversed the glucose-lowering effects of CHs, but only when coadministered with glucose. CHs administered 45 min before the glucose load potentiated the glucose-stimulated insulin secretion. This potentiating effect on insulin secretion was not reversed by the pretreatment with Ex9, it appeared to be enhanced. These results suggest that CHs improve glucose tolerance by inhibiting intestinal glucose uptake and enhancing insulin secretion, and also demonstrated that GLP-1 was partially involved in the inhibition of glucose uptake, but not essential for the enhancement of insulin secretion.

Molecular Cloning, Sequencing, and Expression in Escherichia coli of the Gene Encoding a Novel 5-Oxoprolinase without ATP-Hydrolyzing Activity from Alcaligenes faecalis N-38A

ABSTRACT The gene encoding a novel 5-oxoprolinase without ATP-hydrolyzing activity from Alcaligenes faecalis N-38A was cloned and characterized. The coding region of this gene is 1,299 bp long. The predicted primary protein is composed of 433 amino acid residues, with a 31-amino-acid signal peptide. The mature protein is composed of 402 amino acid residues with a molecular mass of 46,163 Da. The derived amino acid sequence of the enzyme showed no significant sequence similarity to any other proteins reported so far. The 5-oxoprolinase gene was expressed in Escherichia coli by using a regulatory expression system with an isopropyl-β-d-thiogalactopyranoside-inducibletac promoter, and its expression level was approximately 16 mg per liter. The purified enzyme has the same characteristics as the authentic enzyme, except for the amino terminus, which has three additional amino acids. The enzyme was markedly inhibited byp-chloromercuribenzoic acid, EDTA,o-phenanthroline, HgCl2, and CuSO4. The EDTA-inactivated enzyme was completely restored by the addition of Zn2+ or Co2+. In addition, the enzyme was found to contain 1 g-atom of zinc per mol of protein. These results suggest that the 5-oxoprolinase produced by A. faecalis N-38A is a zinc metalloenzyme.

Site-selective chemical modification of chymotrypsin using peptidyl derivatives bearing optically active diphenyl 1-amino-2-phenylethylphosphonate: Stereochemical effect of the diphenyl phosphonate moiety

Diphenyl (α‐aminoalkyl)phosphonates act as mechanism‐based inhibitors against serine proteases by forming a covalent bond with the hydroxy group of the active center Ser residue. Because the covalent bond was found to be broken and replaced by 2‐pyridinaldoxime methiodide (2PAM), we employed a peptidyl derivative bearing diphenyl 1‐amino‐2‐phenylethylphosphonate moiety (Phep(OPh)2) to target the active site of chymotrypsin and to selectively anchor to Lys175 in the vicinity of the active site. Previously, it was reported that the configuration of the α‐carbon of phosphorus in diphenyl (α‐aminoalkyl)phosphonates affects the inactivation reaction of serine proteases, i.e., the (R)‐enantiomeric diphenyl phosphonate is comparable to l‐amino acids and it effectively reacts with serine proteases, whereas the (S)‐enantiomeric form does not. In this study, we evaluated the stereochemical effect of the phosphonate moiety on the selective chemical modification. Epimeric dipeptidyl derivatives, Ala‐(R or S)‐Phep(OPh)2, were prepared by separation with RP‐HPLC. A tripeptidyl (R)‐epimer (Ala‐Ala‐(R)‐Phep(OPh)2) exhibited a more potent inactivation ability against chymotrypsin than the (S)‐epimer. The enzyme inactivated by the (R)‐epimer was more effectively reactivated with 2PAM than the enzyme inactivated by the (S)‐epimer. Finally, N‐succinimidyl (NHS) active ester derivatives, NHS‐Suc‐Ala‐Ala‐ (R or S)‐Phep(OPh)2, were prepared, and we evaluated their action when modifying Lys175 in chymotrypsin. We demonstrated that the epimeric NHS derivative that possessed the diphenyl phosphonate moiety with the (R)‐configuration effectively modified Lys175 in chymotrypsin, whereas that with the (S)‐configuration did not. These results demonstrate the utility of peptidyl derivatives that bear an optically active diphenyl phosphonate moiety as affinity labeling probes in protein bioconjugation.

Altered gene expression profiles associated with enhanced skin inflammation induced by 12-O-tetradecanoylphorbol-13-acetate in streptozotocin-diabetic mice

To examine the mechanisms of diabetes-enhanced inflammation, ear inflammation was induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in streptozotocin (STZ)-injected diabetic and control mice. The inflammatory response was determined from ear thickness and histology. The mRNA expression of several inflammation-related genes 8, 24 and 32 h after TPA treatment was determined by quantitative real-time RT-PCR. Ear thickness did not differ between the two groups at 8 h, but was greater in the diabetic mice than control mice at 24 and 32 h (late phase). STZ-diabetic conditions variously affected TPA-induced gene expression. The changes 8 h after TPA treatment probably reflected transcriptional regulation, and the genes were divided into three groups, up-regulated (IL-6, MCP-1, HO-1 and SOCS3), unregulated (IL-1beta, TNF-alpha and IL-10) and down-regulated (RANTES) genes. TPA-induced gene expression of cytokines, except for RANTES, peaked at 8 h and significantly declined in the late phase in control mice, while the expression of IL-1beta and TNF-alpha did not decline in the late phase in the diabetic mice. This result indicated the destabilization process for these mRNA, a type of post-transcriptional regulation, to be impaired under STZ-induced diabetic conditions; however, TPA-induced gene and protein expression of TTP, an RNA-binding protein involved in mRNA decay, were adversely enhanced in the diabetic mice. These findings suggested that STZ-induced diabetes affected the transcriptional and post-transcriptional control of TPA-induced inflammation, and greater mRNA levels of IL-1beta and TNF-alpha in the late phase were probably responsible for the diabetes-enhanced inflammation.

Visualization of mitochondria in living cells with a genetically encoded yellow fluorescent protein originating from a yellow-emitting luminous bacterium.

We have visualized redox and structural changes in the mitochondria of yeast Saccharomyces cerevisiae as a eukaryotic cell model using a genetically encoded yellow fluorescent protein (Y1-Yellow) and conventional fluorescence microscopy. Y1-Yellow originating from a yellow emitting luminous bacterium Aliivibrio sifiae Y1 was fused with a mitochondria-targeted sequence (mt-sequence). Y1-Yellow fluorescence arising only from the mitochondrial site and the color of yellow fluorescence could be easily differentiated from cellular autofluorescence and from that of conventional probes. Y1-Yellow expressing S. cerevisiae made the yellow fluorescence conspicuous at the mitochondrial site in response to reactive oxygen species (ROS) transiently derived in the wake of pretreatment with hydrogen peroxide. Based on our observation with Y1-Yellow fluorescence, we also showed that mitochondria rearrange to form a cluster structure surrounding chromosomal DNA via respiratory inhibition by cyanide, followed by the generation of ROS. In contrast, uptake of an uncoupler of oxidative phosphorylation is not responsible for mitochondrial rearrangement. These results indicate the utility of Y1-Yellow for visualization of mitochondrial vitality and morphology in living cells.