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K Soda

Publications and source records attributed to K Soda.

At least 55 records · Page 3Linked to original sources
PubMed1999

Stereoisomers of glutathione: preparation and enzymatic reactivities.

We synthesized a series of stereoisomers of glutathione (GSH) and glutathione disulfide (GSSG) by the solid-phase method. These peptides were used to examine their reactivities with enzymes acting on glutathione. The glutathione reductase of yeast acted only on LL-GSSG. Glutathione S-transferase catalyzed the conjugation of 1-chloro-2,4-dinitrobenzene with LL-GSH and DL-GSH (Km (mM): for LL-GSH, 0.035; and for DL-GSH, 0.62), but the DD- and LD-diastereomers were inert. gamma-Glutamyl transpeptidase catalyzed the transfer of gamma-glutamyl moiety of LL-GSH and DL-GSH to taurine forming gamma-glutamyl taurine and cysteinyl taurine (Km (mM): for LL-GSH, 0.336; and for DL-GSH, 0.628), but the other diastereomers were not the substrates. The occurrence of L-cysteinyl residue in the tripeptides is required for the glutathione analogue to be a substrate of the enzymes.

Chromatography, High Pressure Liquid↗
PubMed1999

[Epidemiological study on HIV/AIDS in Cambodia seroprevalence of HIV/STD among commercial sex workers].

OBJECTIVE: To describe epidemiological features of HIV prevalence among female commercial sex workers (CSWs) in Cambodia, a cross-sectional study using a questionnaire study and serological tests was carried out from December 1997 to January 1998. We report the main results of the analyses of serological tests in this article. METHODS: Two hundred ninety six CSWs working in Sisophon and Poi Pet, located in northwest Cambodia, Bantey Mean Chey province, were recruited for interview based on a questionnaire on sexual behavior, and serological tests. The blood samples were examined for HIV antibody, Chlamydia trachomatis IgG antibody, TPHA, Hepatitis B surface antigen, and Hepatitis B surface antibody. The relationship between HIV and the other STD's was analyzed by using logistic regression analysis. RESULTS: The HIV seroprevalence rate was 43.9% (130 out of 296). The seropositive rate of Chlamydia trachomatis IgG antibody (C.T.-IgG-Ab) was 73.3% (217 out of 296). Logistic regression analysis showed a significant association between C.T.-IgG-Ab positive and HIV prevalence. (Odds Ratio: 5.33; 95% Confidence Interval, 2.82-10.07). CONCLUSIONS: This study suggests that the existence of Chlamydia trachomatis is closely related with HIV prevalence among CSWs in Cambodia. Other STDs may also increase susceptibility to male-to-female sexual transmission of HIV. This suggests that appropriate prevention against STDs will be needed for the control of HIV prevalence in Cambodia.

Acquired Immunodeficiency Syndrome↗
PubMed1999

[New aspects of vitamin and coenzyme research].

Vitamins are defined as essential organic micronutrients that are not synthesized by mammals. Coenzymes are defined as organic compounds with low molecular weight that are required to show enzyme activities by reversibly binding with their apoenzymes. Most of vitamins and coenzymes show various biofunctions besides their functions as vitamins and coenzymes. Accordingly, it is more appropriate to understand both as effective biofactors. Various retinoid derivatives synthesized bind to retinoid binding proteins to regulate gene expressions and show other biofunctions. Pyridoxal phosphate serves as an inhibitor of catepsins, and regulates the gene expression. Several novel built-in coenzymes such as topaquinone and lysyltyrosylquinone have been demonstrated in mammalian and microbial enzymes.

Animals↗
PubMed1998

Transamination as a side-reaction catalyzed by alanine racemase of Bacillus stearothermophilus.

The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate, D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4'-3H]pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose epsilon-amino group forms a Schiff base with the C4' aldehyde of pyridoxal 5'-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions. These results suggest that the epsilon-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme.

Alanine Racemase↗
PubMed1998

Reaction mechanism of fluoroacetate dehalogenase from Moraxella sp. B.

Fluoroacetate dehalogenase (EC 3.8.1.3) catalyzes the dehalogenation of fluoroacetate and other haloacetates. The amino acid sequence of fluoroacetate dehalogenase from Moraxella sp. B is similar to that of haloalkane dehalogenase (EC 3.8.1.5) from Xanthobacter autotrophicus GJ10 in the regions around Asp-105 and His-272, which correspond to the active site nucleophile Asp-124 and the base catalyst His-289 of the haloalkane dehalogenase, respectively (Krooshof, G. H., Kwant, E. M., Damborský, J., Koca, J., and Janssen, D. B. (1997) Biochemistry 36, 9571-9580). After multiple turnovers of the fluoroacetate dehalogenase reaction in H218O, the enzyme was digested with trypsin, and the molecular masses of the peptide fragments formed were measured by ion-spray mass spectrometry. Two 18O atoms were shown to be incorporated into the octapeptide, Phe-99-Arg-106. Tandem mass spectrometric analysis of this peptide revealed that Asp-105 was labeled with two 18O atoms. These results indicate that Asp-105 acts as a nucleophile to attack the alpha-carbon of the substrate, leading to the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule on the carbonyl carbon atom. A His-272 --> Asn mutant (H272N) showed no activity with either fluoroacetate or chloroacetate. However, ion-spray mass spectrometry revealed that the H272N mutant enzyme was covalently alkylated with the substrate. The reaction of the H272N mutant enzyme with [14C]chloroacetate also showed the incorporation of radioactivity into the enzyme. These results suggest that His-272 probably acts as a base catalyst for the hydrolysis of the covalent ester intermediate.

Acetates↗
PubMed1998

Twin helical undulator beamline for soft X-ray spectroscopy at SPring-8.

A very high resolution soft X-ray beamline, BL25SU, has been designed and is under construction at SPring-8. Completely right or left circularly polarized light is supplied on a common axis of a newly designed twin helical undulator. A helicity modulation up to 10 Hz can be performed using five kicker magnets. The fundamental radiation covers the region 0.5-3 keV. Higher-order radiation is rather weak on the axis. A monochromator with varied-line-spacing plane gratings is installed to cover the region below 1.5 keV. A very high resolution beyond 10(4) is expected for the whole energy region.

Journal Article↗
PubMed1998

Substrate inhibition of D-amino acid transaminase and protection by salts and by reduced nicotinamide adenine dinucleotide: isolation and initial characterization of a pyridoxo intermediate related to inactivation.

D-Amino acid transaminase, a pyridoxal phosphate (PLP) enzyme, is inactivated by its natural substrate, D-alanine, concomitant with its alpha-decarboxylation [Martinez del Pozo, A., Yoshimura, T., Bhatia, M. B., Futaki, S., Manning, J. M., Ringe, D., and Soda, K. (1992) Biochemistry 31, 6018-6023; Bhatia, M. B., Martinez del Pozo, A., Ringe, D., Yoshimura, T., Soda, K., and Manning, J. M. (1993) J. Biol. Chem. 268, 17687-17694]. beta-Decarboxylation of d-aspartate to d-alanine leads also to this inactivation [Jones, W. M., van Ophem, P. W., Pospischil, M. A., Ringe, D., Petsko, G., Soda, K., and Manning, J. M. (1996) Protein Sci. 5, 2545-2551]. Using a high-performance liquid chromatography-based method for the determination of pyridoxo cofactors, we detected a new intermediate closely related to the inactivation by d-alanine; its formation occurred at the same rate as the inactivation and upon reactivation it reverted to PLP. Conditions were found under which it was characterized by ultraviolet-visible spectral analysis and mass spectroscopy; it is a pyridoxamine phosphate-like compound with a C2 fragment derived from the substrate attached to the C'-4 of the pyridinium ring and it has a molecular mass of 306 consistent with this structure. In the presence of d-serine, slow accumulation of a quinonoid intermediate is also related to inactivation. The inactivation can be prevented by salts, which possibly stabilize the protonated aldimine coenzyme complex. The reduced cofactor, nicotinamide adenine dinucleotide, prevents D-aspartate-induced inactivation. Both of these events also are related to formation of the novel intermediate.

Alanine↗
PubMed1998

Nonstereospecific transamination catalyzed by pyridoxal phosphate-dependent amino acid racemases of broad substrate specificity.

Pyridoxal 5'-phosphate-dependent amino acid racemases of broad substrate specificity catalyze transamination as a side reaction. We studied the stereospecificities for hydrogen abstraction from C-4' of the bound pyridoxamine 5'-phosphate during transamination from pyridoxamine 5'-phosphate to pyruvate catalyzed by three amino acid racemases of broad substrate specificity. When the enzymes were incubated with (4'S)- or (4'R)-[4'-3H]pyridoxamine 5'-phosphate in the presence of pyruvate, tritium was released into the solvent from both pyridoxamine 5'-phosphates. Thus, these enzymes abstract a hydrogen nonstereospecifically from C-4' of the coenzyme in contrast to the other pyridoxal 5'-phosphate-dependent enzymes so far studied, which catalyze the stereospecific hydrogen removal. Amino acid racemase of broad substrate specificity from Pseudomonas putida produced D- and L-glutamate from alpha-ketoglutarate through the transamination with L-ornithine. Because glutamate does not serve as a substrate for racemization, the enzyme catalyzed the nonstereospecific overall transamination between L-ornithine and alpha-ketoglutarate. The cleavage and formation of the C-H bond at C-4' of the coenzyme and C-2 of the substrate thus occurs nonstereospecifically on both sides of the plane of the coenzyme-substrate complex intermediate. Amino acid racemase of broad substrate specificity is the first example of a pyridoxal enzyme catalyzing nonstereospecific transamination.

Amino Acid Isomerases↗
PubMed1998

Properties of glutamate racemase from Bacillus subtilis IFO 3336 producing poly-gamma-glutamate.

We found glutamate racemase activity in cell extracts of Bacillus subtilis IFO 3336, which abundantly produces poly-gamma-glutamate. The highest activity was obtained in the early stationary phase of growth. The racemase was purified to homogeneity. The enzyme was a monomer with a molecular mass of about 30 kDa and required no cofactor. It almost exclusively catalyzed the racemization of glutamate; other amino acids, including alanine and aspartate but not homocysteinesulfinate, were inactive as either substrates or inhibitors. Although the Vmax value of the enzyme for L-glutamate is 21-fold higher than that for D-glutamate, the Vmax/Km value for L-glutamate is almost equal to that for the D-enantiomer. The racemase gene, glr, was cloned into Escherichia coli cells and sequenced. The racemase was overproduced in the soluble fraction of the E. coli clone cells with the substitution of ATG for TTG, the initial codon of the glr gene. D-Amino acid aminotransferase activity was not detected in Bacillus subtilis IFO 3336 cells. B. subtilis CU741, a leuC7 derivative of B. subtilis 168, showed lower glutamate racemase activity and lower productivity of poly-gamma-glutamate than B. subtilis IFO 3336. These results suggest that the glutamate racemase is mainly concerned in D-glutamate synthesis for poly-gamma-glutamate production in B. subtilis IFO 3336.

Amino Acid Isomerases↗
PubMed1998

Construction and properties of a fragmentary D-amino acid aminotransferase.

D-Amino acid aminotransferase [EC 2.6.1.21] catalyzes the inter-conversion between various D-amino acids and alpha-keto acids. The subunit of the homodimeric enzyme from Bacillus sp. YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669]. We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain. When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced. When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity. However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments. The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by alpha-ketoglutarate, and thermostability. The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme.

Alanine Transaminase↗
PubMed1998

A mutant D-amino acid aminotransferase with broad substrate specificity: construction by replacement of the interdomain loop Pro119-Arg120-Pro121 by Gly-Gly-Gly.

D-amino acid aminotransferase (EC 2.6.1.21) catalyzes the interconversion of various D-amino acids and 2-oxo acids. Each homodimer subunit consists of two domains, which are connected by a single loop, Asn118-Pro119-Arg120-Pro121. The loop has no direct contact with the active site region or the cofactor, pyridoxal 5'-phosphate. We attempted to increase the conformational flexibility of this loop through a triple glycine substitution. The resultant mutant P119G-R120G-P121G has features clearly different from the wild-type enzyme under overall as well as half-reaction conditions. The pre-steady-state kinetic analyses of half reactions showed that the mutant enzyme has kmax values higher than the wild-type enzyme towards most D-amino acids examined. A concomitant decrease in substrate affinity (1/Kd), particularly for acidic amino acids, was also observed. A putative binding site for the distal carboxyl group of acidic amino acids in the wild-type enzyme was incidentally displaced by the loop mutation, indicating a functional linkage between the interdomain loop and the active site region. This study has exemplified the usefulness of engineering relatively distant loops as a means to modify substrate specificity of an enzyme.

Amino Acid Substitution↗
PubMed1998

A cold-adapted lipase of an Alaskan psychrotroph, Pseudomonas sp. strain B11-1: gene cloning and enzyme purification and characterization.

A psychrotrophic bacterium producing a cold-adapted lipase upon growth at low temperatures was isolated from Alaskan soil and identified as a Pseudomonas strain. The lipase gene (lipP) was cloned from the strain and sequenced. The amino acid sequence deduced from the nucleotide sequence of the gene (924 bp) corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. LipP also has consensus motifs conserved in other cold-adapted lipases, i.e., Lipase 2 from Antarctic Moraxella TA144 (G. Feller, M. Thirty, J. L. Arpigny, and C. Gerday, DNA Cell Biol. 10:381-388, 1991) and the mammalian hormone-sensitive lipase (D. Langin, H. Laurell, L. S. Holst, P. Belfrage, and C. Holm, Proc. Natl. Acad. Sci. USA 90:4897-4901, 1993): a pentapeptide, GDSAG, containing the putative active-site serine and an HG dipeptide. LipP was purified from an extract of recombinant Escherichia coli C600 cells harboring a plasmid coding for the lipP gene. The enzyme showed a 1,3-positional specificity toward triolein. p-Nitrophenyl esters of fatty acids with short to medium chains (C4 and C6) served as good substrates. The enzyme was stable between pH 6 and 9, and the optimal pH for the enzymatic hydrolysis of tributyrin was around 8. The activation energies for the hydrolysis of p-nitrophenyl butyrate and p-nitrophenyl laurate were determined to be 11.2 and 7.7 kcal/mol, respectively, in the temperature range 5 to 35 degrees C. The enzyme was unstable at temperatures higher than 45 degrees C. The Km of the enzyme for p-nitrophenyl butyrate increased with increases in the assay temperature. The enzyme was strongly inhibited by Zn2+, Cu2+, Fe3+, and Hg2+ but was not affected by phenylmethylsulfonyl fluoride and bisnitrophenyl phosphate. Various water-miscible organic solvents, such as methanol and dimethyl sulfoxide, at concentrations of 0 to 30% (vol/vol) activated the enzyme.

Cloning, Molecular↗
PubMed1998

Purification, characterization, and mechanism of a flavin mononucleotide-dependent 2-nitropropane dioxygenase from Neurospora crassa.

A nitroalkane-oxidizing enzyme was purified to homogeneity from Neurospora crassa. The enzyme is composed of two subunits; the molecular weight of each subunit is approximately 40,000. The enzyme catalyzes the oxidation of nitroalkanes to produce the corresponding carbonyl compounds. It acts on 2-nitropropane better than on nitroethane and 1-nitropropane, and anionic forms of nitroalkanes are much better substrates than are neutral forms. The enzyme does not act on aromatic compounds. When the enzyme reaction was conducted in an 18O2 atmosphere with the anionic form of 2-nitropropane as the substrate, acetone (with a molecular mass of 60 Da) was produced. This indicates that the oxygen atom of acetone was derived from molecular oxygen, not from water; hence, the enzyme is an oxygenase. The reaction stoichiometry was 2CH3CH(NO2)CH3 + O2-->2CH3COCH3 + 2HNO2, which is identical to that of the reaction of 2-nitropropane dioxygenase from Hansenula mrakii. The reaction of the Neurospora enzyme was inhibited by superoxide anion scavengers in the same manner as that of the Hansenula enzyme. Both of these enzymes are flavoenzymes; however, the Neurospora enzyme contains flavin mononucleotide as a prosthetic group, whereas the Hansenula enzyme contains flavin adenine dinucleotide.

Acetone↗
PubMed1998

Endo-beta-glucanase secreted by a psychrotrophic yeast: purification and characterization.

A psychrotrophic yeast, Rhodotorula glutinis KUJ 2731, isolated from soil, effectively produced an extracellular endo-beta-glucanase (EC 3.2.1.4). The enzyme was monomeric, and the molecular mass was about 40,000 Da. The N-terminal amino acid sequence was H-Ser-Leu-Pro- Lys-Leu-Gly-Gly-Val-Asp-Leu-Ala-Gly-Leu-Asp-Ile-Gly-Lys-Asp-Lys-Asn-. alpha-Helix content was calculated to be about 32.6%. The isoelectric point was 8.57. The activation energy was 20.9 kJ/mol, which was much smaller than that of mesophilic enzymes. The enzyme was active at temperatures from 0 to 70 degrees C, with a highest initial velocity at 50 degrees C similar to other psychrotrophic enzymes. The enzyme was inhibited by Hg2+. The enzyme catalyzed hydrolysis of carboxymethyl cellulose with an apparent K(m) of 1.1% and Vmax of 556 mumol/min/mg. Products from the enzymatic hydrolysis of carboxymethyl cellulose by the enzyme were glucose, cellobiose, and cellotriose. The enzyme also catalyzed the transglycosylation of p-nitrophenyl-beta-cellotrioside to cellotetraose.

Amino Acid Sequence↗
PubMed1998

Compensation for D-glutamate auxotrophy of Escherichia coli WM335 by D-amino acid aminotransferase gene and regulation of murI expression.

D-glutamate, an indispensable component of peptidoglycans of bacteria, is provided by glutamate racemase in E. coli cells. Compensation for D-glutamate auxotrophy of E. coli WM335 cells lacking the glutamate racemase gene, murI, with the D-amino acid aminotransferase gene suggests that presence of a threshold concentration for the D-glutamate required by E. coli cells, as well as a regulation system for murI expression.

Alanine Transaminase↗