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K Soda

Publications and source records attributed to K Soda.

At least 199 records · Page 11Linked to original sources
PubMed1990

Substitution of S-(beta-aminoethyl)-cysteine for active-site lysine of thermostable aspartate aminotransferase.

The active site lysyl residue (K239) of the thermostable aspartate aminotransferase [EC 2.6.1.1] was replaced by cysteinyl residue by means of site-directed mutagenesis. The K239C mutant enzyme obtained was catalytically inactive. The reaction of the cysteinyl residue of the K239C mutant enzyme with ethylenimine led to the formation of S-(beta-aminoethylcysteinyl (SAEC) residue. The K239SAEC mutant enzyme obtained showed about 25% of the activity of wild-type enzyme, and absorbed at 375 nm, which suggested the internal Schiff base formation.

Amino Acids↗
PubMed1990

Thermostable alanine racemase. Its structural stability.

The gene encoding thermostable alanine recemase from Bacillus stearothermophilus was cloned and expressed in E. coli. The enzyme was purified to homogeneity from cell extracts of E. coli carrying a plasmid designated pICR4. The alanine racemase gene sequenced was found to contain an open reading frame of 1158 nucleotides. The molecular weight of the enzyme subunit was estimated to be 43,341. The alpha-helical and beta-structure contents were calculated to be about 34 and 26%, respectively, from CD data. CD measurements of the denaturation process of enzyme by guanidine hydrochloride showed the presence of a stable intermediate during the denaturation. Limited proteolysis with subtilisin resulted in the formation of two dissimilar peptide fragments with molecular weights of about 28,000 and 13,000 in the early stage of the digestion. These suggest that the enzyme subunit is composed of two structurally dissimilar domains connected by a short polypeptide (residues 258-266), which first suffers the limited proteolysis. However, the enzyme retained almost full activity and the conformation indistinguishable from the intact protein even when it was proteolytically hydrolyzed to more than 10 fragments.

Alanine Racemase↗
PubMed1990

Purification and characterization of thermostable aspartate aminotransferase from a thermophilic Bacillus species.

Aspartate aminotransferase (EC 2.6.1.1) was purified to homogeneity from cell extracts of a newly isolated thermophilic bacterium, Bacillus sp. strain YM-2. The enzyme consisted of two subunits identical in molecular weight (Mr, 42,000) and showed microheterogeneity, giving two bands with pIs of 4.1 and 4.5 upon isoelectric focusing. The enzyme contained 1 mol of pyridoxal 5'-phosphate per mol of subunit and exhibited maxima at about 360 and 415 nm in absorption and circular dichroism spectra. The intensities of the two bands were dependent on the buffer pH; at neutral or slightly alkaline pH, where the enzyme showed its maximum activity, the absorption peak at 360 nm was prominent. The enzyme was specific for L-aspartate and L-cysteine sulfinate as amino donors and alpha-ketoglutarate as an amino acceptor; the KmS were determined to be 3.0 mM for L-aspartate and 2.6 mM for alpha-ketoglutarate. The enzyme was most active at 70 degrees C and had a higher thermostability than the enzyme from Escherichia coli. The N-terminal amino acid sequence (24 residues) did not show any similarity with the sequences of mammalian and E. coli enzymes, but several residues were identical with those of the thermoacidophilic archaebacterial enzyme recently reported.

Amino Acid Sequence↗
PubMed1990

Purification and characterization of S-alkylcysteine alpha,beta-lyase from Pseudomonas putida.

S-Alkylcysteine alpha,beta-lyase [EC 4.4.1.6] was purified to more than 90% homogeneity from the cell extract of Pseudomonas putida ICR 3640. The enzyme has a molecular weight of about 195,000, and is composed of six subunits identical in molecular weight (37,000). Pyridoxal 5'-phosphate is required as a cofactor. The enzyme catalyzes the alpha,beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, Se-methyl-DL-selenocysteine, and O-methyl-L-serine. However, S-methyl-D-cysteine, L-methionine, and L-norvaline were inert. The enzyme catalyzes also the beta-replacement reaction of the thiomethyl group of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-DL-selenocysteine and O-methyl-L-serine also serve as substrates in the beta-replacement reaction.

Amino Acids↗
PubMed1990

Thermostable S-alkylcysteine alpha, beta-lyase from a thermophile: purification and properties.

S-Alkylcysteine alpha, beta-lyase was found in a thermophile, Bacillus sp. 41A, which was newly isolated from soil, and purified to homogeneity from the cell extract. The enzyme has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (39,000). The enzyme requires pyridoxal 5'-phosphate as a coenzyme, and catalyzes alpha, beta-elimination of S-methyl-L-cysteine and its analogs such as S-ethyl-L-cysteine, L-djenkolate, L-cystine, Se-methyl-L-selenocysteine, and O-methyl-DL-serine. However, S-methyl-D-cysteine, D-cystine, L-methionine, and L-norleucine were inert. The enzyme also catalyzes the beta-replacement reaction of S-methyl-L-cysteine with various thiols to yield the corresponding S-substituted cysteines. In addition to S-methyl-L-cysteine, Se-methyl-L-selenocysteine and O-methyl-DL-serine also serve as beta-substituent acceptors in the beta-replacement reaction. The enzyme is most active at 70 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 44 amino acids. The amino acid sequence in the vicinity of the lysyl residue to which pyridoxal 5'-phosphate is bound, was -Lys-His-Gln-Arg- by Edman degradation of the pyridoxyl peptide obtained by digestion with trypsin after reduction with sodium borohydride.

Amino Acid Sequence↗
PubMed1990

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase.

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.

Amino Acid Isomerases↗
PubMed1989

Effects of D-serine on bacterial D-amino acid transaminase: accumulation of an intermediate and inactivation of the enzyme.

Incubation of pure bacterial D-amino acid transaminase with D-serine or erythro-beta-hydroxy-DL-aspartic acid, which are relatively poor substrates, leads to generation of a new absorbance band at 493 nm that is probably the quinonoid intermediate. The 420-nm absorbance band (due to the pyridoxal phosphate coenzyme) decreases, and the 338-nm absorbance band (due to the pyridoxamine phosphate or some other form of the coenzyme) increases. A negative Cotton effect at 493 nm in the circular dichroism spectra is also generated. Closely related D amino acids do not lead to generation of this new absorption band, which has a half-life of the order of several hours. Treatment of the enzyme with the good substrate D-alanine leads to a small but detectable amount of the same absorbance band. D-Serine but not erythro-beta-hydroxyaspartate leads to inactivation of D-amino acid transaminase, and D-alanine affords partial protection. The results indicate that D-serine is a unique type of inhibitor in which the initial steps of the half-reaction of transamination are so slow that a quinonoid intermediate with a 493-nm absorption band accumulates. A derivative formed from this intermediate inactivates the enzyme.

Circular Dichroism↗
PubMed1989

Stereospecificity of reactions catalyzed by bacterial D-amino acid transaminase.

The spectral shift from 420 to 338 nm when pure bacterial D-amino acid transaminase binds D-amino acid substrates is also exhibited in part by high concentrations of L-amino acids (L-alanine and L-glutamate) but not by simple dicarboxylic acids or monoamines. Slow processing of L-alanine to D-alanine was observed both by coupled enzymatic assays using D-amino acid oxidase and by high pressure liquid chromatography analysis employing an optically active chromophore (Marfey's reagent). When the acceptor for L-alanine was alpha-ketoglutarate, D-glutamate was also formed. This minor activity of the transaminase involved both homologous (L-alanine and D-alanine) and heterologous (L-alanine and D-glutamate) substrate pairs and was a function of the nature of the keto acid acceptor. In the presence of alpha-ketoisovalerate, DL-alanine was almost completely processed to D-valine; within the limits of the assay no L-valine was detected. With alpha-ketoisocaproate, 90% of the DL-alanine was converted to D-leucine. In the mechanism of this transaminase reaction, there may be more stereoselective constraints for the protonation of the quinonoid intermediate during the second half-reaction of the transamination reaction, i.e. the donation of the amino group from the pyridoxamine 5'-phosphate coenzyme to a second keto acid acceptor, than during removal of the alpha proton in the initial steps of the reaction pathway. Thus, with this D-amino acid transaminase, the discrete steps of transamination ensure fidelity of the stereospecificity of reaction pathway.

Bacillus↗
PubMed1989

Enzymatic in situ determination of stereospecificity of NAD-dependent dehydrogenases.

Amino acid racemases inherently catalyze the exchange of alpha-hydrogen of amino acids with deuterium during racemization in 2H2O. When the reactions catalyzed by alanine racemase (EC 5.1.1.1) and L-alanine dehydrogenase (EC 1.4.1.1), which is pro-R specific for the C-4 hydrogen transfer of NADH, are coupled in 2H2O, [4R-2H]NADH is exclusively produced. Similarly, [4S-2H]NADH is made in 2H2O with amino-acid racemase with low substrate specificity (EC 5.1.1.10) and L-leucine dehydrogenase (EC 1.4.1.9), which is pro-S specific. We have established a simple procedure for the in situ analysis of stereospecificity of C-4 hydrogen transfer of NADH by an NAD-dependent dehydrogenase by combination with either of the above two couples of enzymes in the same reaction mixture. When the C-4 hydrogen of NAD+ is fully retained after sufficient incubation, the stereospecificity of hydrogen transfer by a dehydrogenase is the same as that of alanine dehydrogenase or leucine dehydrogenase. However, when the C-4 hydrogen of NAD+ is exchanged with deuterium, the enzyme to be examined shows the different stereospecificity from alanine dehydrogenase or leucine dehydrogenase. Thus, we can readily determine the stereospecificity by 1H NMR measurement without isolation of the coenzymes and products.

Alanine Dehydrogenase↗
PubMed1989

The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases.

The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli. The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product. The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226. Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E. coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H. (1985) J. Biochem. (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size. Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar. The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene.

Amino Acid Sequence↗
PubMed1989

Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species. Purification, characterization, and active site sequence determination.

D-Amino acid aminotransferase was found in several thermophilic Bacillus species and purified to homogeneity from the best producer, Bacillus sp. YM-1, which was newly isolated from a sauna dust. The enzyme has a molecular weight of about 62,000 and consists of two subunits identical in molecular weight (30,000). It catalyzes transamination between various D-amino acids and alpha-keto acids, although the substrate specificity is narrower than the enzyme from the mesophile, Bacillus sphaericus (Yonaha, K., Misono, H., Yamamoto, T., and Soda, K. (1975) J. Biol. Chem. 250, 6983-6989). The Bacillus sp. YM-1 enzyme is most active at 60 degrees C and stable at high temperatures. Automated Edman degradation provided the N-terminal sequence of the first 20 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 3 amino acids. The amino acid sequence in the vicinity of the lysyl residue, Lys(Pxy), that binds pyridoxal 5'-phosphate was determined as Cys-Asp-Ile-Lys(Pxy)-Ser-Leu-Asn-Leu-Leu-Gly-Ala-Val-Leu-Ala-Lys- from the pyridoxyl peptide obtained by digestion with trypsin. The active site sequence is markedly different from those of L-amino acid aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes.

Amino Acid Sequence↗
PubMed1989

Activity and spectroscopic properties of bacterial D-amino acid transaminase after multiple site-directed mutagenesis of a single tryptophan residue.

One of the three tryptophan residues per subunit of thermostable D-amino acid transaminase, Trp-139, is close to the active-site Lys-145 in the sequence of the protein. This tryptophan has been changed to several other types of residues by site-directed mutagenesis. The only mutant protein that was sufficiently active and stable for study had Phe substituted for Trp (W139F). The spectroscopic properties of this mutant enzyme differed from those of the wild-type transaminase. For example, denatured W139F showed the expected decrease in fluorescence emission intensity at 350 nm due to the deletion of one Trp residue, but the fluorescence emission of the wild-type and W139F enzymes in the native state did not differ in intensity. This result suggests that the fluorescence of Trp-139 in the native, wild-type enzyme is not manifested perhaps due to its proximity to the coenzyme, pyridoxal phosphate. Results of energy-transfer studies at several wavelengths could also be interpreted as due to the proximity of Trp-139 and the coenzyme. Circular dichroism studies indicated that the negative Cotton effect at 420 nm due to the coenzyme was still present in W139F. However, the 280-nm optically active band present in the wild-type enzyme was greatly diminished in W139F. The mutant protein with Asp at position 139 (W139D) could not be isolated presumably because it was degraded. The other mutant enzymes, W139P, W139A, and W139H, were isolated with partial activities (15-35%) that were slowly lost upon storage at 4 degrees C. Overall, these results indicate the importance of Trp-139 in the thermostable D-amino acid transaminase.

Bacillus↗
PubMed1989

A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).

Amino Acid Sequence↗
PubMed1989

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization.

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min. The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.

Alanine Dehydrogenase↗
PubMed1989

Enzymatic in situ analysis by 1H-NMR of the hydrogen transfer stereospecificity of NAD(P)+-dependent dehydrogenases.

We have established a simple procedure for the in situ analysis of stereospecificity of an NAD(P)-dependent dehydrogenase for C-4 hydrogen transfer of NAD(P)H by means of glutamate racemase [EC 5.1.13] and glutamate dehydrogenase [EC 1.4.1.3]. Glutamate racemase inherently catalyzes the exchange of alpha-H of glutamate with 2H during racemization in 2H2O. When the reactions of glutamate racemase and glutamate dehydrogenase, which is pro-S specific for the C4-H transfer of NAD(P)H, are coupled in 2H2O, [4S-2H]-NAD(P)H is exclusively produced. Therefore, if 1H is fully retained at C-4 of NAD(P)+ after incubation of a reaction mixture containing both the enzymes and a dehydrogenase to be tested, the stereospecificity of the dehydrogenase is the same as that of glutamate dehydrogenase. When the C4-H of NAD(P)+ is exchanged with 2H, the enzyme to be examined is different from glutamate dehydrogenase in stereospecificity. Thus, we can readily determine the stereospecificity by 1H-NMR measurement of NAD(P)+ without isolation of the coenzymes and products.

Amino Acid Isomerases↗
PubMed1989

HLA-DQw3 in Japanese summer-type hypersensitivity pneumonitis induced by Trichosporon cutaneum.

The phenotype of the major histocompatibility complex, loci A, B, C, DR, and DQ, was studied in 66 patients with Japanese summer-type hypersensitivity pneumonitis induced by Trichosporon cutaneum, and the results were compared with those obtained from 472 normal healthy subjects. Our results showed an increase in the frequency of HLA-DQw3 antigen in the patients (66% compared with 49% in the normal healthy control subjects: chi 2 = 5.48, p = 0.018, RR = 1.92), suggesting the importance of the host factors for the generation of this disease. The association between HLA-DQw3 as an immune suppression gene and the disease is discussed.

Alveolitis, Extrinsic Allergic↗