Biomedical subjects

Kiyoshi Hayashi

Publications and source records attributed to Kiyoshi Hayashi.

At least 19 recordsLinked to original sources

PubMed2006

Structural dissection of the reaction mechanism of cellobiose phosphorylase.

Cellobiose phosphorylase, a member of the glycoside hydrolase family 94, catalyses the reversible phosphorolysis of cellobiose into alpha-D-glucose 1-phosphate and D-glucose with inversion of the anomeric configuration. The substrate specificity and reaction mechanism of cellobiose phosphorylase from Cellvibrio gilvus have been investigated in detail. We have determined the crystal structure of the glucose-sulphate and glucose-phosphate complexes of this enzyme at a maximal resolution of 2.0 A (1 A=0.1 nm). The phosphate ion is strongly held through several hydrogen bonds, and the configuration appears to be suitable for direct nucleophilic attack to an anomeric centre. Structural features around the sugar-donor and sugar-acceptor sites were consistent with the results of extensive kinetic studies. When we compared this structure with that of homologous chitobiose phosphorylase, we identified key residues for substrate discrimination between glucose and N-acetylglucosamine in both the sugar-donor and sugar-acceptor sites. We found that the active site pocket of cellobiose phosphorylase was covered by an additional loop, indicating that some conformational change is required upon substrate binding. Information on the three-dimensional structure of cellobiose phosphorylase will facilitate engineering of this enzyme, the application of which to practical oligosaccharide synthesis has already been established.

Binding Sites↗

PubMed2006

RAISE: a simple and novel method of generating random insertion and deletion mutations.

Although proteins may be artificially improved by random insertion and deletion mutagenesis methods, these procedures are technically difficult, and the mutations introduced are no more variable than those introduced by the introduction of random point mutations. We describe here a three-step method called RAISE, which is based on gene shuffling and can introduce a wide variety of insertions, deletions and substitutions. To test the efficacy of this method, we used it to mutate TEM beta-lactamase to generate improved antibiotic resistance. Some unique insertion or deletion mutations were observed in the improved mutants, some of which caused higher activities than point mutations. Our findings indicate that the RAISE method can yield unique mutants and may be a powerful technique of protein engineering.

Amino Acid Substitution↗

PubMed2006

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose).

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.

Calcium Gluconate↗

PubMed2005

Co-refolding of two peptide fragments derived from Agrobacterium tumefaciens beta-glucosidase with catalytic activity.

Four sites of the non-homologous region (coding amino acid residues of 347, 421, 466 and 533) of a gene were randomly selected for splitting to investigate the function of beta-glucosidase from Agrobacterium tumefaciens in the co-refolding of peptides into the catalytically active enzyme. As a result of gene splitting, four N- and C-terminal domain peptides were obtained as insoluble inclusion bodies. No catalytic activity was observed when these fragments refolded individually. However, a considerable amount of activity was restored when the two fragments derived from N- and C- terminal peptides were co-refolded together. The deletion of amino acid residues in the non-homologous region resulted in a complete loss of enzyme activity, which suggests that truncation of amino acids in this region strongly affects the co-refolding ability of the enzyme to maintain activity.

Agrobacterium tumefaciens↗

PubMed2005

A single amino acid substitution enhances the catalytic activity of family 11 xylanase at alkaline pH.

Random mutagenesis of the gene encoding family 11 xylanase was used to obtain alkalophilic mutants. The catalytic domain of the chimeric enzyme Stx15, which was constructed from Streptomyces lividans xylanase B and Thermobifida fusca xylanase A, was mutated using error-prone PCR and screened for halo formation on dye-linked xylan plates and activity toward soluble xylan. A positive mutant, M1011, was isolated, and it was found that mutation A49V was responsible for the alkalophilicity of the mutant. Mutation A49V increased the specific activity at pH 9.1 and the stability of mutant A49V was not significantly different from that of Stx15 at 60 degrees C. Both enzymes retained more than 90% of their relative activity from pH 4.7 to 9.1 after 1 h of incubation at 60 degrees C. Analysis of the kinetic parameters at various pH values showed that the A49V mutation reduced the Km in the alkaline pH range, resulting in the higher specific activity of the A49V mutant enzyme.

Amino Acid Sequence↗

PubMed2005

The role of conserved arginine residue in loop 4 of glycoside hydrolase family 10 xylanases.

An arginine residue in loop 4 connecting beta strand 4 and alpha-helix 4 is conserved in glycoside hydrolase family 10 (GH10) xylanases. The arginine residues, Arg(204) in xylanase A from Bacillus halodurans C-125 (XynA) and Arg(196) in xylanase B from Clostridium stercorarium F9 (XynB), were replaced by glutamic acid, lysine, or glutamine residues (XynA R204E, K and Q, and XynB R196E, K and Q). The pH-k(cat)/K(m) and the pH-k(cat) relationships of these mutant enzymes were measured. The pK(e2) and pK(es2) values calculated from these curves were 8.59 and 8.29 (R204E), 8.59 and 8.10 (R204K), 8.61 and 8.19 (R204Q), 7.42 and 7.19 (R196E), 7.49 and 7.18 (R196K), and 7.86 and 7.38 (R196Q) respectively. Only the pK(es2) value of arginine derivatives was less than those of the wild types (8.49 and 9.39 [XynA] and 7.62 and 7.82 [XynB]). These results suggest that the conserved arginine residue in GH10 xylanases increases the pK(a) value of the proton donor Glu during substrate binding. The arginine residue is considered to clamp the proton donor and subsite +1 to prevent structural change during substrate binding.

Amino Acid Sequence↗

PubMed2004

Novel PCR-mediated mutagenesis employing DNA containing a natural abasic site as a template and translesional Taq DNA polymerase.

We describe a novel method of PCR-mediated mutagenesis employing DNA containing a natural abasic site and translesional Taq DNA polymerase. This method incorporated an adenine (80.8%) or guanine (7.7%) residue or led to a base deletion mutation (11.2%) opposite the abasic site. We conclude that the combination of DNA containing an abasic site and translesional Taq DNA polymerase is an easy and useful technique for PCR-mediated mutagenesis, having advantages different from those of conventional error-prone PCR.

DNA↗

PubMed2004

One-step random mutagenesis by error-prone rolling circle amplification.

In vitro random mutagenesis is a powerful tool for altering properties of enzymes. We describe here a novel random mutagenesis method using rolling circle amplification, named error-prone RCA. This method consists of only one DNA amplification step followed by transformation of the host strain, without treatment with any restriction enzymes or DNA ligases, and results in a randomly mutated plasmid library with 3-4 mutations per kilobase. Specific primers or special equipment, such as a thermal-cycler, are not required. This method permits rapid preparation of randomly mutated plasmid libraries, enabling random mutagenesis to become a more commonly used technique.

Ceftazidime↗

PubMed2004

Transglycosylation reaction of xylanase B from the hyperthermophilic Thermotoga maritima with the ability of synthesis of tertiary alkyl beta-D-xylobiosides and xylosides.

The recombinant xylanase B (XynB) of Thermotoga maritima MSB8 was characterized and was found to cleave p-nitrophenyl beta-D-xyloside via the transglycosylation reaction in the previous study. XynB was activated in the presence of alcohols, and XynB activity was increased by iso-propanol (2M) to 2.1-fold. This type of activation was investigated and was shown to be due to the transglycosylation activity with p-nitrophenyl beta-D-xylobioside being converted to alkyl beta-D-xylobiosides in the presence of XynB and alcohols. Through the transglycosylation reaction, alkyl beta-xylosides and xylobiosides were simultaneously produced in the presence of xylan and alcohols. Primary alcohols were found to be the best acceptors. The highest yields of alkyl beta-xylosides and xylobiosides were 33% and 50% of the total sugar, respectively. XynB showed a great ability to transfer xylose and xylobiose to secondary alcohol acceptors, and was unique for being able to synthesize the tertiary alkyl beta-xylosides and xylobiosides with high yields of 18.2% and 11.6% of the total sugar, respectively. This is the first report of a xylanase with the ability to synthesize tertiary alkyl beta-xylosides and xylobiosides. The specificity of the beta-linkage was confirmed by the proton nuclear magnetic resonance ((1)H NMR). Thus, XynB of T. maritima appears to be an ideal enzyme for the synthesis of useful alkyl beta-xylosides and xylobiosides.

Alkylation↗

PubMed2004

Crystallization and preliminary X-ray analysis of cellobiose phosphorylase from Cellvibrio gilvus.

A recombinant cellobiose phosphorylase from Cellvibrio gilvus has been prepared and crystallized by the sitting-drop vapour-diffusion method using 10 mg ml(-1) purified enzyme, 1.5 M ammonium sulfate, 0.1 M MES buffer pH 7.0 and 5 mM glucose. A suitable crystal was obtained after 10 d incubation at 298 K. The crystal belongs to space group P2(1), with unit-cell parameters a = 84.77, b = 98.31, c = 104.04 A, beta = 102.73 degrees. X-ray diffraction data to 2.1 A resolution have been collected at KEK-PF BL-5A.

Cellvibrio↗

PubMed2004

Kinetic evidence related to substrate-assisted catalysis of family 18 chitinases.

The hydrolytic reaction of family 18 chitinase has been considered to occur via substrate assisted catalysis. To kinetically investigate the enzyme reaction mechanism, we synthesized compounds designed to reduce the polarization of the carbonyl in N-acetyl group, GlcNAc-GlcN(TFA)-UMB (2) and GlcNAc-GlcN(TAc)-UMB (3). Kinetic parameters in the hydrolysis of these compounds by chitinase A from Serratia marcescens (ChiA) were compared with those from the hydrolysis of (GlcNAc)2-UMB (1). The kcat of 2 was 3.4% of 1, but the Km of 2 was 10-fold that of 1. In contrast, the kcat of 3 was only 0.3% of that of 1, and the two reactions had an identical Km. The drastic decreases in kcat were probably due to the weak nucleophilic activity of the C2-N-trifluoroacetamide and N-thioacetamide groups at reducing ends of compounds 2 and 3, respectively. These results indicate that the anchimeric assistance of the C2 N-acetamide group at GlcNAc plays a key role in the hydrolytic reactions catalyzed by family 18 chitinases.

Acetylation↗

PubMed2004

N-myristoyltransferase inhibitor protein is homologous to heat shock cognate protein 70.

Many of viral and eukaryotic proteins are required for signal transduction and regulatory functions which undergo a lipid modification by the enzyme N-myristoyltransferase (NMT). In this study, we demonstrated that heat shock cognate protein 70 (HSC70) is homologous to NMT inhibitor protein (NIP71), which was discovered in our laboratory, based on MALDI-TOF mass spectrometric analysis. The purified bovine cytosolic HSC70 and particulate NIP71 produced a dose-dependent inhibition of human NMT having half maximal inhibitions of 235 and 230 nM, respectively. Further, Western blot analysis revealed that the purified particulate NIP71 and cytosolic HSC70 cross-reacted with both anti-NIP71 and anti-HSC70 antibodies. The results we obtained imply that molecular chaperones could be involved in the regulation of NMT in normal and cancerous cells. Further studies directed to revealing the role of HSC70 in the regulation of NMT may lead to the development of gene based therapies of colon cancer.

Acyltransferases↗

PubMed2004

Purification and characterization of an intracellular cycloalternan-degrading enzyme from Bacillus sp. NRRL B-21195.

A novel intracellular cycloalternan-degrading enzyme (CADE) was purified to homogeneity from the cell pellet of Bacillus sp. NRRL B-21195. The enzyme has a molecular mass of 125 kDa on SDS-PAGE. The pH optimum was 7.0, and the enzyme was stable from pH 6.0 to 9.2. The temperature optimum was 35 degrees C and the enzyme exhibited stability up to 50 degrees C. The enzyme hydrolyzed cycloalternan [CA; cyclo(-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(-->3)-alpha-d-Glcp-(1-->)] as the best substrate, to produce only isomaltose via an intermediate, alpha-isomaltosyl-(1-->3)-isomaltose. This enzyme also hydrolyzed isomaltosyl substrates, such as panose, alpha-isomaltosyl-(1-->4)-maltooligosaccharides, alpha-isomaltosyl-(1-->3)-glucose, and alpha-isomaltosyl-(1-->3)-isomaltose to liberate isomaltose. Neither maltooligosaccharides nor isomaltooligosaccharides were hydrolyzed by the enzyme, indicating that CADE requires alpha-isomaltosyl residues connected with (1-->4)- or (1-->3)-linkages. The K(m) value of cycloalternan (1.68 mM) was 20% of that of panose (8.23 mM). The k(cat) value on panose (14.4s(-1)) was not significantly different from that of cycloalternan (10.8 s(-1)). Judging from its specificity, the systematic name of the enzyme should be cycloalternan isomaltosylhydrolase. This intracellular enzyme is apparently involved in the metabolism of starch via cycloalternan in Bacillus sp. NRRL B-21195, its role being to hydrolyze cycloalternan inside the cells.

Bacillus↗

PubMed2004

Crystallization and preliminary X-ray analysis of xylanase B from Clostridium stercorarium.

Recombinant mature xylanase B from Clostridium stercorarium has been prepared and crystallized by the sitting-drop vapour-diffusion method using 4 mg ml(-1) purified enzyme, 10.3%(w/v) polyethylene glycol 1500, 8.6%(v/v) glycerol and 0.34 M non-detergent sulfobetaine 195. A suitable crystal grew after incubation for ten weeks at 293 K. The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.76, b = 96.60, c = 138.44 A. X-ray diffraction data were collected to 1.80 A resolution.

Clostridium↗

PubMed2004

Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio.

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-alpha-D-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with alpha-D-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce beta-D-glucosyl-(1-->4)-2-acetamide-2-deoxy-D-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-beta-glycosides of GlcNAc as the acceptor substrate with 10-20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k(0)=5.5 s(-1), K(m)=2.0 mM; synthetic reaction, k(0)=10 s(-1), K(m)=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

Amino Acid Sequence↗

PubMed2004

Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold.

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.

Amino Acid Sequence↗

PubMed2004

Analyses of PCR products using DNA templates containing a consecutive deoxyinosine sequence.

We determined nucleotide incorporation specificity of four thermostable DNA polymerases opposite deoxyinosine in order to evaluate the mutagenic effect of the presence of a consecutive deoxyinosine sequence during PCR amplification. Deoxycytosine was the only incorporated nucleotide opposite deoxyinosine during PCR amplification, irrespective of origin of DNA polymerase.

Base Pairing↗

PubMed2004

Effects of truncation at the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens.

The function of the non-homologous region of a family 3 beta-glucosidase from Agrobacterium tumefaciens (Cbg1) was studied by analyzing the properties of mutant enzymes that have internal truncated amino acid sequences in the region. Five truncated mutants named Cbg1-d4, Cbg1-d31, Cbg1-d62, Cbg1-d89, and Cbg1-d119 having deletions of 4, 31, 62, 89, and 119 amino acid residues starting from Phe417, respectively, were expressed in Escherichia coli and purified. All the mutants exhibited beta-glucosidase activity, indicating that the non-homologous region was not essential for the activity. The truncation caused thermal instability, decrease in pK(a) of the proton donor residue (Glu616), and deficient transglycosylation activity. The thermal stability and the pK(a) of Glu616 were partially recovered with longer truncation, suggesting that the truncation perturbed the structure and that their presence in the region was not essential. The main role of the non-homologous region could be formation of a hydrophobic atmosphere at the acceptor site to make the enzyme suitable for hydrolyzing hydrophobic glucosides.

Agrobacterium tumefaciens↗