Biomedical subjects

N Esaki

Publications and source records attributed to N Esaki.

At least 73 records · Page 4Linked to original sources

PubMed1996

Stereospecificity of thermostable ornithine 5-aminotransferase for the hydrogen transfer in the L- and D-ornithine transamination.

The thermostable ornithine 5-aminotransferase of a thermophile, Bacillus sp. YM-2, is unique in acting on both enantiomers of ornithine, although less effectively on the D-enantiomer. We studied the stereospecificity of the enzyme for the hydrogen abstraction from C-5 of the substrate moiety and the addition and removal of the hydrogen at C-4' of the cofactor (pyridoxal phosphate and pyridoxamine phosphate) moiety of the external Schiff base intermediate in the transamination of L- and D-ornithine. L- and D-[5-3H]ornithines were prepared by incubation of L- and D-ornithines with the enzyme in 3H2O, respectively. When the L-[5-3H]ornithine was incubated with L-ornithine 5-aminotransferase of a mesophile, Bacillus sphaericus, which catalyzes the stereospecific abstraction of pro-S hydrogen from C-5 of L-ornithine, most of the tritium was released into the solvent. The D-[5-3H]ornithine also reacted with the enzyme of B. sphaericus in the presence or absence of the amino acid racemase of Pseudomonas putida. Tritium was released only in the presence of the racemase, which catalyzes the racemization of ornithine but does not act on C-5 of ornithine. These results show that the Bacillus sp. YM-2 ornithine 5-aminotransferase stereospecifically abstracts the pro-S hydrogen from C-5 of L- and D-ornithine. When the apo form of the enzyme was incubated with pyridoxamine 5'-phosphate that was stereospecifically tritiated at C-4' and 2-oxoglutarate in the presence of L-ornithine or D-ornithine, tritium was released exclusively from (4'S)-[4'-3H]pyridoxamine. Therefore, addition and abstraction of hydrogen at C-4' of the cofactor moiety stereospecifically occur on the si face of the external Schiff base intermediate in the overall transamination catalyzed by Bacillus sp. YM-2 ornithine 5-aminotransferase irrespective of the C-2 configuration of the amino donor.

Amino Acid Isomerases↗

PubMed1996

Crystallization and preliminary x-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp. YL.

The dimeric L-2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 angstrom, b = 62.78 angstrom, c = 50.84 angstrom, and beta = 122.4 degrees, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 angstrom resolution.

Crystallography, X-Ray↗

PubMed1996

[Trace elements as activation factors of enzymes: characteristic enzymes and their activation mechanisms].

We here describe activation mechanisms of three characteristic metalloenzymes by metals. The copper-containing amine oxidase contains a topaquinone residue at the active site. The inactive precursor form of the enzyme, which contains a tyrosine residue at the position corresponding to topaquinone of the matured protein, was isolated and converted to the matured form by incubation with cupric ions through self-processing of the protein. The selenocysteine residue of selenium-containing formate dehydrogenase is synthesized cotranslationally through a process directed by a UGA codon that normally functions as a stop codon. We describe the mechanism of recognition of UGA and specific incorporation of selenium into the protein. Vanadium-containing haloperoxidases afford stable apo forms lacking vanadium. Reconstitution of holo forms by vanadium ions are also described.

Amine Oxidase (Copper-Containing)↗

PubMed1995

Reaction mechanism of L-2-haloacid dehalogenase of Pseudomonas sp. YL. Identification of Asp10 as the active site nucleophile by 18O incorporation experiments.

L-2-Haloacid dehalogenase (EC 3.8.1.2) catalyzes the hydrolytic dehalogenation of L-2-haloacids to produce the corresponding D-2-hydroxy acids. We have analyzed the reaction mechanism of the enzyme from Pseudomonas sp. YL and found that Asp10 is the active site nucleophile. When the multiple turnover enzyme reaction was carried out in H2(18)O with L-2-chloropropionate as a substrate, lactate produced was labeled with 18O. However, when the single turnover enzyme reaction was carried out by use of a large excess of the enzyme, the product was not labeled. This suggests that an oxygen atom of the solvent water is first incorporated into the enzyme and then transferred to the product. After the multiple turnover reaction in H2(18)O, the enzyme was digested with lysyl endopeptidase, and the molecular masses of the peptide fragments formed were measured by an ionspray mass spectrometer. Two 18O atoms were shown to be incorporated into a hexapeptide, Gly6-Lys11. Tandem mass spectrometric analysis of this peptide revealed that Asp10 was labeled with two 18O atoms. Our previous site-directed mutagenesis experiment showed that the replacement of Asp10 led to a significant loss in the enzyme activity. These results indicate that Asp10 acts as a nucleophile on the alpha-carbon of the substrate leading to the formation of an ester intermediate, which is hydrolyzed by nucleophilic attack of a water molecule on the carbonyl carbon atom.

Amino Acid Sequence↗

PubMed1995

Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9.

A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43,000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermophilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Oshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056-9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity; the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.

Amino Acid Oxidoreductases↗

PubMed1995

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10.

The gene of NAD(+)-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.

Amino Acid Sequence↗

PubMed1995

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL.

The gene encoding thermostable L-2-halo acid dehalogenase of Pseudomonas sp. YL was isolated, and its overexpression system was constructed. Gene library was prepared from Sau3AI fragments of total DNA from Ps. sp. YL, pUC118 as a vector and Escherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.

Cloning, Molecular↗

PubMed1995

In vivo effect of GroESL on the folding of glutamate racemase of Escherichia coli.

The overexpression of the murI (glr) gene, which encodes the glutamate racemase of Escherichia coli, resulted in the formation of inclusion bodies of the enzyme, and little activity was found in the soluble fraction of the transformant cells. The coexpression of the groESL gene with murI caused an in vivo solubilization of glutamate racemase in an active form. We isolated the active enzyme and purified it effectively.

Amino Acid Isomerases↗

PubMed1995

Role of leucine 201 of thermostable D-amino acid aminotransferase from a thermophile, Bacillus sp. YM-1.

We studied the catalytic role of leucine 201 residue of the thermostable D-amino acid aminotransferase: the residue was shown crystallographically to be in the vicinity of the active site to interact with the bound pyridoxal phosphate. We replaced the leucine 201 by alanyl or tryptophanyl residues by means of site-directed mutagenesis. The L201A and L201W mutant enzymes showed anomalous kinetic behavior in the overall reaction. The reaction rates of the L201A and L201W mutant enzymes gradually decreased with an increase in the reaction time to become practically zero at a high concentration of substrates. The mutant enzymes were also inactivated in the half reaction with D-alanine, although more slowly than in the overall reaction. The absorption spectra of the mutant enzymes in the presence of D-alanine and alpha-ketoglutarate suggest that the enzyme molecules were mostly in the pyridoxamine form under the conditions employed. These phenomena were explained by assuming two (or more) enzyme species showing kinetically different catalysis for pyridoxamine form of the mutant enzymes, and the rate of conversion from one of these pyridoxamine forms to the pyridoxal form should be very low. The leucine 201 residue probably regulates the function of cofactor during the reaction of D-amino acid aminotransferase.

Alanine↗

PubMed1995

Structural analysis of the L-methionine gamma-lyase gene from Pseudomonas putida.

The gene encoding L-methionine gamma-lyase from Pseudomonas putida was cloned and the primary structure of the enzyme was deduced from its nucleotide sequence. The L-methionine gamma-lyase gene was expressed in Escherichia coli. The amino acid sequences of BrCN-digested peptides agreed with the corresponding parts of the L-methionine gamma-lyase sequence determined from the gene structure. The polypeptide is composed of 398 amino acid residues with a calculated molecular weight of 42,626, corresponding to the subunit of the homotetrameric enzyme. The deduced amino acid sequence of L-methionine gamma-lyase only showed extensive homology with other well known alpha,gamma-elimination and/or gamma-replacement pyridoxal 5'-phosphate-dependent enzymes, such as cystathionine gamma-lyase, cystathionine gamma-synthase, and O-acetylhomoserine O-acetylserine sulfhydrylase, that participate in the biosynthesis of sulfur amino acids. However, the deduced essential cysteine residue of L-methionine gamma-lyase was not conserved in these enzymes. We confirmed the presence of a part of an open reading frame in the 3'-flanking region of the L-methionine gamma-lyase gene, which showed high homology with the N-terminal region of pyruvate dehydrogenase (lipoamide) from E. coli, suggesting that it participates in the degradative pathway for L-methionine together with L-methionine gamma-lyase.

Amino Acid Sequence↗

PubMed1995

Comprehensive site-directed mutagenesis of L-2-halo acid dehalogenase to probe catalytic amino acid residues.

L-2-Halo acid dehalogenase catalyzes the stereospecific hydrolytic dehalogenation of L-2-halo acids, with inversion of the C2-configuration. Seven L-2-halo acid dehalogenases from various bacterial strains are significantly similar to one another in their amino acid sequences (36-70% identity), and they are supposed to catalyze the reaction through the same mechanism. To identify catalytically important residues, we mutated all the 36 highly conserved charged and polar amino acid residues of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which consists of 232 amino acid residues, by replacement of D by N, E by Q, R by K, and vice versa, S and T by A, Y and W by F, M by L, and H by N. We found that the replacement of D10, K151, S175, D180, R41, S118, T14, Y157, and N177 led to a significant loss in the enzyme activity or an increase in the Km value for the substrate, showing their involvement in the catalysis. The roles of these residues are discussed.

Amino Acid Sequence↗

PubMed1995

Thermostable ornithine aminotransferase from Bacillus sp. YM-2: purification and characterization.

Thermostable L-ornithine: alpha-ketoglutarate delta-aminotransferase (L-ornithine: 2-oxo-acid 5-aminotransferase) [EC 2.6.1.13] was purified to homogeneity from Bacillus sp. YM-2. The enzyme has a molecular weight of about 82,000 and consists of two subunits with identical molecular weights. The enzyme catalyzes transamination from L-ornithine to alpha-ketoglutarate, producing L-glutamate and L-glutamate gamma-semialdehyde, which is spontaneously dehydrated to L-delta 1-pyrroline-5-carboxylate, and the enzyme is most active at 70 degrees C. In addition to L-ornithine, the enzyme unexpectedly acts on D-ornithine, the reaction rate being 6% of that for L-ornithine. The enzyme contains 1 mol each of pyridoxal 5'-phosphate and another vitamin B6 compound per mol. The enzyme released the bound pyridoxal 5'-phosphate, as judged from the absorption at 425 nm on incubation with 2.0 M guanidine hydrochloride. The resultant inactive enzyme still gave a 340-nm peak and contained 1 mol of the vitamin B6 compound. The partial amino acid sequence shows high homology with those of mammalian and yeast ornithine delta-aminotransferases.

Amino Acid Sequence↗

PubMed1995

Synthesis of fluoroacetate from fluoride, glycerol, and beta-hydroxypyruvate by Streptomyces cattleya.

Streptomyces cattleya produces fluoroacetate and 4-fluorothreonine from inorganic fluoride added to the culture broth. We have shown by 19F nuclear magnetic resonance (NMR) spectrometry that fluoroacetate is accumulated first in the culture broth and that accumulation of 4-fluorothreonine is next. To show precursors of the carbon skeleton of fluoroacetate, we carried out tracer experiments with various 14C- and 13C-labeled compounds. Radioactivity of [U-14C]glucose, [U-14C]glycerol, [U-14C]serine, and [U-14C]beta-hydroxypyruvate was incorporated into fluoroacetate to an extent of 0.2 to 0.4%, whereas [3-14C]pyruvate, [2,3-14C]succinate, and [U-14C]aspartate were less efficiently incorporated (0.04 to 0.08%). The addition of [2-13C]glycerol to the mycelium suspension of Streptomyces cattleya caused exclusive enrichment of the carboxyl carbon of fluoroacetate with 13C; about 40% of carboxyl carbon of fluoroacetate was labeled with 13C. We studied the radioactivity incorporation of [3-14C]-, [U-14C]-, and [1-14C]beta-hydroxypyruvates to show that C-2 and C-3 of beta-hydroxypyruvate are exclusively converted to the carbon skeleton of fluoroacetate. These results suggest that the carbon skeleton of fluoroacetate derives from C-1 and C-2 of glycerol through beta-hydroxypyruvate, whose hydroxyl group is eventually replaced by fluoride.

Carbon Isotopes↗

PubMed1994

Unique primary structure of 2-nitropropane dioxygenase from Hansenula mrakii.

We have isolated the gene encoding 2-nitropropane dioxygenase from Hansenula mrakii, an FAD enzyme that catalyzes the oxygenative denitrification of various anionic nitroalkanes. The gene contained an open reading frame consisting of 1122 nucleotides corresponding to 374 amino acid residues. The protein molecular mass was estimated to be 41,466 Da, which was similar to the subunit molecular mass of the enzyme determined by SDS/PAGE. Several FAD enzymes such as D-amino acid oxidase and glucose oxidase also catalyze the oxidation of nitroalkanes as a side-reaction, although not so efficiently [Kido, T. & Soda, K. (1984) Arch. Biochem. Biophys. 234, 468-475]. However, we found no proteins in the databases (GenBank, EMBL, PIR and SWISS-PROT) which are homologous to 2-nitropropane dioxygenase of H. mrakii in primary structure. No protein motifs, including a nucleotide-binding motif, GXGXXG, were found in PROSITE, a database of biologically significant protein sites and patterns. Accordingly, 2-nitropropane dioxygenase is a new type of flavoprotein with a unique structure.

Amino Acid Sequence↗

PubMed1994

Bacterial glutamate racemase has high sequence similarity with myoglobins and forms an equimolar inactive complex with hemin.

Glutamate racemase (EC 5.1.1.3), an enzyme of microbial origin, shows significant sequence homology with mammalian myoglobins, in particular in the regions corresponding to the E and F helices, which constitute the heme binding pocket of myoglobins. Glutamate racemase binds tightly an equimolar amount of hemin, leading to loss of racemase activity. Although this enzyme shows homology with aspartate racemase, the latter does not bind hemin. The glutamate racemase gene of Pediococcus pentosaceus has a 795-nt open reading frame and encodes 265-amino acid residues, which form a monomeric protein (M(r) 29,000). Neither racemase has cofactors, but they contain essential cysteine residues [Yohda, M., Okada, H. & Kumagai, H. (1991) Biochim. Biophys. Acta 1089, 234-240].

Amino Acid Isomerases↗

PubMed1994

The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius.

Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4 degrees C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70 degrees C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, L-leucine, NADH, 2-oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial-velocity analyses. The enzyme showed pro-S stereospecificity for hydrogen transfer of NADH in the reductive amination. The enzyme gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098-bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (M(r) 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery.

Actinomyces↗

PubMed1994

A simple method for determination of stereospecificity of aminotransferases for C-4' hydrogen transfer of the coenzyme.

A simple method was established for determination of the stereospecificity of C-4' hydrogen transfer of the coenzymes (pyridoxal and pyridoxamine). The method is based on the findings that aspartate aminotransferase of pig heart and D-amino acid aminotransferase of Bacillus sp. YM-1 catalyze the abstraction of the pro-S and pro-R proton at C-4' of pyridoxamine, respectively. Pyridoxal is a poor coenzyme, but readily released from the enzyme. It reacts in 3H2O with a substrate amino acid and an apo-aminotransferase whose stereospecificity for C-4' hydrogen transfer is to be determined. The resultant pyridoxamine which is tritiated at C-4' is incubated with an apo form of aspartate aminotransferase or D-amino acid aminotransferase and a substrate, alpha-keto acid. The stereospecificity for the C-4' hydrogen transfer examined is determined by measurement of radioactivity retained in the pyridoxal formed. We showed by means of this method that C-4' hydrogen transfer of coenzyme occurs on the si face of the external Schiff base in the transamination reactions of two aspartate aminotransferases of Bacillus sp. YM-2 and Escherichia coli, and aromatic amino acid aminotransferase of E. coli.

Alanine Transaminase↗

PubMed1994

Replacement of active-site lysine-239 of thermostable aspartate aminotransferase by S-(2-aminoethyl)cysteine: properties of the mutant enzyme.

The active-site lysine residue of thermostable aspartate aminotransferase, Lys-239, to which the cofactor, pyridoxal 5'-phosphate (PLP), is bound, has been converted to Cys by site-directed mutagenesis. The thiol group of Cys-239 was chemically aminoethylated with ethylenimine. Amino acid analysis of the modified enzyme showed that it contained about 1 mol of S-(2-aminoethyl)cysteine (SAEC) per mol subunit. The activity of the mutant enzyme (K239SAEC) was about 14% of that of the wild-type enzyme. No significant difference in thermostability was found between the wild-type and K239SAEC enzymes. The UV-visible spectrum of K239SAEC showed a peak (lambda max 380 nm), due to absorption by the cofactor, at a 20 nm longer wavelength than that of the wild-type enzyme. The circular dichroism band due to the bound cofactor of K239SAEC also shifted toward a 20 nm longer wavelength. We determined kinetic parameters (rate constants, kmax, and dissociation constants, Kd, for the substrates) for each half transamination catalyzed by the wild-type and K239SAEC mutant enzymes by the stopped-flow method. The kmax values for the mutant enzyme reactions were 2.6-24 times lower than those for the wild-type enzyme ones. The two enzymes showed similar Kd values for the same substrates except glutamate; the mutant enzyme showed higher affinity for glutamate than the wild-type enzyme.

Aspartate Aminotransferases↗