Biomedical subjects

Nobuyoshi Esaki

Publications and source records attributed to Nobuyoshi Esaki.

At least 19 recordsLinked to original sources

PubMed2006

Biochemical and genetic analysis of the gamma-resorcylate (2,6-dihydroxybenzoate) catabolic pathway in Rhizobium sp. strain MTP-10005: identification and functional analysis of its gene cluster.

We identified a gene cluster that is involved in the gamma-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graRDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes gamma-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 +/- 0.82] x 10(-4)) of graF was detected in the total RNA of the cells after one hour of cultivation when gamma-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by gamma-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on gamma-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the gamma-resorcylate catabolic pathway in an aerobic bacterium.

Base Sequence↗

PubMed2006

Enzymatic synthesis of L-pipecolic acid by Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida.

L-Pipecolic acid is a chiral pharmaceutical intermediate. An enzymatic system for the synthesis of L-pipecolic acid from L-lysine by commercial L-lysine alpha-oxidase from Trichoderma viride and an extract of recombinant Escherichia coli cells coexpressing Delta1-piperideine-2-carboxylate reductase from Pseudomonas putida and glucose dehydrogenase from Bacillus subtilis is described. A laboratory-scale process provided 27 g/l of L-pipecolic acid in 99.7% e.e.

Bacillus subtilis↗

PubMed2006

Mechanism of the reaction catalyzed by DL-2-haloacid dehalogenase as determined from kinetic isotope effects.

dl-2-Haloacid dehalogenase from Pseudomonas sp. 113 is a unique enzyme because it acts on the chiral carbons of both enantiomers, although its amino acid sequence is similar only to that of d-2-haloacid dehalogenase from Pseudomonas putida AJ1 that specifically acts on (R)-(+)-2-haloalkanoic acids. Furthermore, the catalyzed dehalogenation proceeds without formation of an ester intermediate; instead, a water molecule directly attacks the alpha-carbon of the 2-haloalkanoic acid. We have studied solvent deuterium and chlorine kinetic isotope effects for both stereoisomeric reactants. We have found that chlorine kinetic isotope effects are different: 1.0105 +/- 0.0001 for (S)-(-)-2-chloropropionate and 1.0082 +/- 0.0005 for the (R)-(+)-isomer. Together with solvent deuterium isotope effects on V(max)/K(M), 0.78 +/- 0.09 for (S)-(-)-2-chloropropionate and 0.90 +/- 0.13 for the (R)-(+)-isomer, these values indicate that in the case of the (R)-(+)-reactant another step preceding the dehalogenation is partly rate-limiting. Under the V(max) conditions, the corresponding solvent deuterium isotope effects are 1.48 +/- 0.10 and 0.87 +/- 0.27, respectively. These results indicate that the overall reaction rates are controlled by different steps in the catalysis of (S)-(-)- and (R)-(+)-reactants.

Hydrolases↗

PubMed2006

Physicochemical and pharmacokinetic characterization of highly potent recombinant L-methionine gamma-lyase conjugated with polyethylene glycol as an antitumor agent.

A highly potent recombinant L-methionine gamma-lyase (METase) conjugated with polyethylene glycol (PEG) was characterized physicochemically and pharmacokinetically in vivo and in vitro. Pegylated METase (PEG-METase), which contains pyridoxal 5'-phosphate (PLP) as a cofactor in the molecule, is a potent anticancer agent that can deplete L-methionine from plasma. Although pegylation decreased its specific activity, dithiothreitol (DTT) treatment increased it over three times with the detachment of one PEG moiety modified with a cysteine residue. We can produce DTT-treated PEG-METase on a large scale in sufficient quality for therapeutic use. The superiority of DTT-treated PEG-METase was confirmed by the enhancement of L-methionine depletion and amelioration of pharmacokinetics in mice. The holoenzyme of DTT-treated PEG-METase gave a several times larger area under the plasma concentration curve than that of DTT-untreated PEG-METase, not because of an increase of the half-life but because of high specific activity. Conversely, simultaneous PLP infusion led to a greatly increased half-life of the holoenzyme. DTT-treated PEG-METase administration with PLP infusion was the most useful combination for maximizing the potency of the enzyme. We showed that serum albumin interfered with holoenzyme activity in vitro. The decrease of holoenzyme activity was dependent on the type of serum albumin. We concluded that PLP was released from PEG-METase by serum albumin in vivo and in vitro. The deleterious effect of PLP dissociation from PEG-METase could be improved by supplementing PLP and oleic acid. Their synergistic effect in preventing a decrease of the holoenzyme activity was also observed.

Animals↗

PubMed2005

Physiological role of D-amino acid-N-acetyltransferase of Saccharomyces cerevisiae: detoxification of D-amino acids.

Saccharomyces cerevisiae is sensitive to D-amino acids: those corresponding to almost all proteinous L-amino acids inhibit the growth of yeast even at low concentrations (e.g. 0.1 mM). We have determined that D-amino acid-N-acetyltransferase (DNT) of the yeast is involved in the detoxification of D-amino acids on the basis of the following findings. When the DNT gene was disrupted, the resulting mutant was far less tolerant to D-amino acids than the wild type. However, when the gene was overexpressed with a vector plasmid p426Gal1 in the wild type or the mutant S. cerevisiae as a host, the recombinant yeast, which was found to show more than 100 times higher DNT activity than the wild type, was much more tolerant to D-amino acids than the wild type. We further confirmed that, upon cultivation with D-phenylalanine, N-acetyl-D-phenylalanine was accumulated in the culture but not in the wild type and hpa3Delta cells overproducing DNT cells. Thus, D-amino acids are toxic to S. cerevisiae but are detoxified with DNT by N-acetylation preceding removal from yeast cells.

Amino Acids↗

PubMed2005

Crystal structures of Delta1-piperideine-2-carboxylate/Delta1-pyrroline-2-carboxylate reductase belonging to a new family of NAD(P)H-dependent oxidoreductases: conformational change, substrate recognition, and stereochemistry of the reaction.

Delta(1)-Piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase from Pseudomonas syringae pv. tomato belongs to a novel sub-class in a large family of NAD(P)H-dependent oxidoreductases distinct from the conventional MDH/LDH superfamily characterized by the Rossmann fold. We have determined the structures of the following three forms of the enzyme: the unliganded form, the complex with NADPH, and the complex with NADPH and pyrrole-2-carboxylate at 1.55-, 1.8-, and 1.7-A resolutions, respectively. The enzyme exists as a dimer, and the subunit consists of three domains; domain I, domain II (NADPH binding domain), and domain III. The core of the NADPH binding domain consists of a seven-stranded predominantly antiparallel beta-sheet fold (which we named SESAS) that is characteristic of the new oxidoreductase family. The enzyme preference for NADPH over NADH is explained by the cofactor binding site architecture. A comparison of the overall structures revealed that the mobile domains I and III change their conformations to produce the catalytic form. This conformational change plays important roles in substrate recognition and the catalytic process. The active site structure of the catalytic form made it possible to identify the catalytic Asp:Ser:His triad and investigate the catalytic mechanism from a stereochemical point of view.

Binding Sites↗

PubMed2005

High-level expression and bulk crystallization of recombinant L-methionine gamma-lyase, an anticancer agent.

L-Methionine gamma-lyase is a pyridoxal 5'-phosphate-dependent enzyme which has tumor selective anticancer activity. An efficient production process for the recombinant enzyme was constructed by using the overexpression plasmid in Escherichia coli, large-scale cultivation, and practical crystallization on an industrial scale. The plasmid was optimized with a promoter and the region of the ribosome-binding site. Plasmid pMGLTrc03, which has a trc promoter and a spacing of 12 nucleotides between the Shine-Dalgarno sequence and the ATG translation initiation codon, was selected as the most suitable plasmid. The transformants produced the enzyme, which intracellularly accumulated at 2.1 mg/ml as an active form and accounted for 43% of the total proteins in the soluble fraction by simple batch fermentation using a 500-l fermentor. The crystals were directly obtained from crude enzyme with 87% yield by a crystallization in the presence of 9.0% polyethylene glycol 6000, 3.6% ammonium sulfate, and 0.18 M sodium chloride using a 100-l crystallizer. After recrystallization, the enzyme was purified by anion-exchange column chromatography to remove endotoxins and by gel filtration for polishing. We prepared 600 g of purified enzyme with a low endotoxin content of sufficient quality for therapeutical use, with a 41% overall yield in the purification process.

Antimetabolites, Antineoplastic↗

PubMed2005

2-Haloacrylate reductase, a novel enzyme of the medium chain dehydrogenase/reductase superfamily that catalyzes the reduction of a carbon-carbon double bond of unsaturated organohalogen compounds.

A soil bacterium, Burkholderia sp. WS, grows on 2-chloroacrylate as the sole carbon source. To identify the enzymes metabolizing 2-chloroacrylate, we carried out comparative two-dimensional gel electrophoresis of the proteins from 2-chloroacrylate- and lactate-grown bacterial cells. As a result, we found that a protein named CAA43 was inducibly synthesized when the cells were grown on 2-chloroacrylate. The CAA43 gene was cloned and shown to encode a protein of 333 amino acid residues (M(r) 35,788) that shared a significant sequence similarity with NADPH-dependent quinone oxidoreductase from Escherichia coli (38.2% identity). CAA43 was overproduced in E. coli and purified to homogeneity. The purified protein catalyzed the NADPH-dependent reduction of the carbon-carbon double bond of 2-chloroacrylate to produce (S)-2-chloropropionate, which is probably further metabolized to (R)-lactate by (S)-2-haloacid dehalogenase in Burkholderia sp. WS. NADH did not serve as a reductant. Despite the sequence similarity to quinone oxidoreductases, CAA43 did not act on 1,4-benzoquinone and 1,4-naphthoquinone. 2-Chloroacrylate analogs, such as acrylate and methacrylate, were also inert as the substrates. In contrast, 2-bromoacrylate served as the substrate. Thus, we named this novel enzyme 2-haloacrylate reductase. This study revealed a new pathway for the degradation of unsaturated organohalogen compounds. It is also notable that the enzyme is useful for the production of (S)-2-chloropropionate, which is used for the industrial production of aryloxyphenoxypropionic acid herbicides.

Acrylates↗

PubMed2005

Site-directed mutagenesis alters DnaK-dependent folding process.

The overproduction of d-aminoacylase (A6-d-ANase) of Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) is accompanied by aggregation of the overproduced protein, and its soluble expression is facilitated by the coexpression of DnaK-DnaJ-GrpE (DnaKJE). When the A6-d-ANase gene was expressed in the Escherichia coli dnaK mutant dnaK756, little activity was observed in the soluble fraction, and it was restored by the coexpression of DnaKJE or the substitution of the R354 residue of A6-d-ANase for lysine. These results suggest that the guanidino group of the R354 residue of A6-d-ANase disturbs its proper folding in the absence of DnaK and the disturbance is eliminated by binding of DnaK to the R354 residue in the presence of DnaK. This is the first report that the DnaK-dependent folding process of the enzyme is altered by site-directed mutagenesis.

Alcaligenes↗

PubMed2005

N-methyl-L-amino acid dehydrogenase from Pseudomonas putida. A novel member of an unusual NAD(P)-dependent oxidoreductase superfamily.

We found N-methyl-L-amino acid dehydrogenase activity in various bacterial strains, such as Pseudomonas putida and Bacillus alvei, and cloned the gene from P. putida ATCC12633 into Escherichia coli. The enzyme purified to homogeneity from recombinant E. coli catalyzed the NADPH-dependent formation of N-alkyl-L-amino acids from the corresponding alpha-oxo acids (e.g. pyruvate, phenylpyruvate, and hydroxypyruvate) and alkylamines (e.g. methylamine, ethylamine, and propylamine). Ammonia was inert as a substrate, and the enzyme was clearly distinct from conventional NAD(P)-dependent amino acid dehydrogenases, such as alanine dehydrogenase (EC 1.4.1.1). NADPH was more than 300 times more efficient than NADH as a hydrogen donor in the enzymatic reductive amination. Primary structure analysis revealed that the enzyme belongs to a new NAD(P)-dependent oxidoreductase superfamily, the members of which show no sequence homology to conventional NAD(P)-dependent amino acid dehydrogenases and opine dehydrogenases.

Chromatography, Gel↗

PubMed2005

DnaK from Vibrio proteolyticus: complementation of a dnaK-null mutant of Escherichia coli and the role of its ATPase domain.

We cloned the 4.8-kbp DNA fragment containing the dnaK gene from the chromosomal DNA of Vibrio proteolyticus. It contained four genes arranged unidirectionally in the order of grpE, gltP, dnaK and dnaJ. The DnaK gene of V. proteolyticus (VprDnaK) allowed a dnaK-null mutant of Escherichia coli (DeltadnaK52) to propagate lambda phages but not to grow at 43 degrees C. However, a chimeric DnaK gene comprising the regions corresponding to the N-terminal ATPase domain of E. coli DnaK (EcoDnaK) and the C-terminal region of VprDnaK including the substrate-binding domain, enabled the mutant to grow at 43 degrees C. The temperature dependence for the ATPase activity of the chimeric DnaK was similar to that of EcoDnaK. Fluorometric analyses showed that the chimeric DnaK is much more thermostable than EcoDnaK and VprDnaK. These findings indicate that the thermal stability of the ATPase domain of DnaK is responsible for its chaperone action at high temperatures such as 43 degrees C.

Adenosine Triphosphatases↗

PubMed2005

A new family of NAD(P)H-dependent oxidoreductases distinct from conventional Rossmann-fold proteins.

A new family of NAD(P)H-dependent oxidoreductases is now recognized as a protein family distinct from conventional Rossmann-fold proteins. Numerous putative proteins belonging to the family have been annotated as malate dehydrogenase (MDH) or lactate dehydrogenase (LDH) according to the previous classification as type-2 malate/L-lactate dehydrogenases. However, recent biochemical and genetic studies have revealed that the protein family consists of a wide variety of enzymes with unique catalytic activities other than MDH or LDH activity. Based on their sequence homologies and plausible functions, the family proteins can be grouped into eight clades. This classification would be useful for reliable functional annotation of the new family of NAD(P)H-dependent oxidoreductases.

Amino Acid Sequence↗

PubMed2004

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures.

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0 degrees C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24 degrees C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43 degrees C nor lambda phage propagation at an even lower temperature, 30 degrees C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15 degrees C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.

Adenosine Triphosphatases↗

PubMed2004

The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent delta1-piperideine-2-carboxylate/delta1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline.

A Pseudomonas putida ATCC12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.

Bacterial Proteins↗

PubMed2004

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.

Animals↗

PubMed2004

Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B.

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon-fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the alpha-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.

Amino Acid Substitution↗

PubMed2004

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability.

The gene encoding an esterase (PsyEst) of Psychrobacter sp. Ant300, a psychrophilic bacterium isolated from Antarctic soil, was cloned, sequenced, and expressed in Escherichia coli. PsyEst, which is a member of hormone-sensitive lipase (HSL) group of the lipase/esterase family, is a cold-active, themolabile enzyme with high catalytic activity at low temperatures (5-25 degrees C), low activation energy (e.g., 4.6 kcal/mol for hydrolysis of p-nitrophenyl butyrate), and a t(1/2) value of 16 min for thermal inactivation during incubation at 40 degrees C and pH 7.9. A three-dimensional structural model of PsyEst predicted that Gly(244) was located in the loop near the active site of PsyEst and that substitution of this amino-acid residue by proline should potentially rigidify the active-site environment of the enzyme. Thus, we introduced the Gly(244)-->Pro substitution into the enzyme. Stability studies showed that the t(1/2) value for thermal inactivation of the mutant during incubation at 40 degrees C and pH 7.9 was 11.6 h, which was significantly greater than that of the wild-type enzyme. The k(cat)/K(m) value of the mutant was lower for all substrates examined than the value of the wild type. Moreover, this amino-acid substitution caused a shift of the acyl-chain length specificity of the enzyme toward higher preference for short-chain fatty acid esters. All of these observations could be explained in terms of a decrease in active-site flexibility brought about by the mutation and were consistent with the hypothesis that cold activity and thermolability arise from local flexibility around the active site of the enzyme.

Amino Acid Sequence↗

PubMed2004

D-amino acid N-acetyltransferase of Saccharomyces cerevisiae: a close homologue of histone acetyltransferase Hpa2p acting exclusively on free D-amino acids.

D-Amino acid N-acetyltransferase is a unique enzyme of Saccharomyces cerevisiae acting specifically on D-amino acids. The enzyme was found to be encoded by HPA3, a putative histone/protein acetyl transferase gene, and we purified its gene product, Hpa3p, from recombinant Escherichia coli cells. Hpa3p shares 49% sequence identity and 81% sequence similarity with a histone acetyltransferase, Hpa2p, of S. cerevisiae. Hpa3p acts on a wide range of D-amino acids but shows extremely low activity toward histone. However, Hpa2p does not act on any of the free amino acids except L-lysine and D-lysine. Kinetic analyses suggest that Hpa3p catalyzes the N-acetylation of D-amino acids through an ordered bi-bi mechanism, in which acetyl-CoA is the first substrate to be bound and CoA is the last product to be liberated.

Acetyltransferases↗