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K Soda

Publications and source records attributed to K Soda.

At least 127 records · Page 7Linked to original sources
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

Determination of free D-amino acids with a bacterial transaminase: their depletion leads to inhibition of bacterial growth.

A general procedure is described to determine the common free D-amino acids except D-proline in mixtures that also contain L-amino acids. The system employs exogenous pure bacterial D-amino acid transaminase coupled with 2-oxohexanoate, which accepts the amino group from D-amino acids to form D-norleucine. This amino acid is readily quantified by amino acid analysis since it elutes in a position not occupied by any of the common amino acids. Formation of norleucine denotes the presence of some D-amino acid(s) whose identity can be established by a corresponding decrease in the susceptible amino acid(s) after treatment. The utility of the procedure is demonstrated by determination of the amounts of free D-alanine and free D-glutamate in extracts of Escherichia coli JM-103 grown on minimal medium; D-alanine was the major D-amino acid. By the same principle, 2-oxohexanoate through coupling with endogenous bacterial D-amino acid transaminase is shown to be capable of inhibiting the growth of E. coli by depleting it of the D-alanine and D-glutamate.

Amino Acids↗
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

Serum lidocaine and MEGX concentrations after pharyngeal anesthesia for gastroscopy.

In 20 patients undergoing topical anesthesia for gastroscopic examination, serum concentrations of lidocaine and its metabolite, monomethylglycinexylidide (MEGX), were measured. Patients were divided randomly into two groups: a gel group, in which 5 ml of 2% lidocaine gel was applied to the throat for 20 minutes; and a solution group, in which 40 ml of 2% lidocaine solution was administered by gargling for five minutes. The effect of oropharyngeal anesthesia was comparable in both groups. In the gel group, the serum levels of lidocaine and MEGX were not elevated at 15 minutes after application of the anesthetic. However, in the solution group, a rise in both serum lidocaine and MEGX at 15 minutes after anesthesia was detected in some of the patients (40%). Increased serum MEGX concentrations, which correlated well with serum lidocaine concentrations, were associated with the age of the patient (r = 0.606; p < 0.05), but not with height or weight. As a topical anesthetic for endoscopic examination, we prefer lidocaine gel to lidocaine solution, because the latter might be absorbed more rapidly and unpredictably in some, especially aged patients.

Adult↗
PubMed1994

A newly developed fine-caliber endoscope for peroral cholangiopancreatoscopy.

We have developed a new fine-caliber (2.09 mm outer diameter) endoscope for peroral cholangiopancreatoscopy. The endoscope contains one image transmission fiber, 12 light guide fibers (the transmitter of light from the light source) and a working channel (a lumen for the guide wire and rinsing). The working channel, whose bore is 0.72 mm, is located centrally within the endoscope. The endoscope can be introduced reliably into the bile and pancreatic ducts using the same techniques as those for endoscopic nasobiliary drainage through the instrumental channel of a duodenoscope for examination without pretreatment of the papilla of Vater. Two patients with lesions of the pancreatic duct and seven patients with lesions of the bile duct suspected or detected by endoscopic retrograde cholangiopancreatography (ERCP) were examined. Direct inspection of the biliopancreatic duct not only provided enough information to make a definite diagnosis, but also revealed lesions that were not detectable by ERCP or other examinations.

Adult↗
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↗
PubMed1994

Studies of the active-site lysyl residue of thermostable aspartate aminotransferase: combination of site-directed mutagenesis and chemical modification.

The gene technological substitution of the cysteinyl residue for the pyridoxal 5'-phosphate-binding lysyl residue (K239) of thermostable aspartate aminotransferase of Bacillus sp. YM-2 led to loss of the activity of the enzyme, which inherently contains no cysteinyl residues. The cysteinyl residue of the mutant enzyme was modified to lysine sulfur analog residues, S-(beta-aminoethyl)cysteine (SAEC), S-(beta-aminopropyl)cysteine (SAPC), and S-(beta-aminoethylthio)cysteine (SATC) with 2-bromoethylamine, 3-bromopropylamine, and 2-mercaptoethylamine, respectively. The modified mutant enzymes showed absorbance at 379 (K239SAEC), 400 (K239SAPC), and 365 nm (K239SATC), whereas the spectrum of the wild-type enzyme exhibited an absorption maximum at 360 nm derived from the internal Schiff base at pH 8.0. The absorption of the modified mutant enzymes at these wavelengths disappeared on reduction with NaCNBH3. This suggests that omega-amino groups of the introduced lysine sulfur analog residue form an internal Schiff base with pyridoxal 5'-phosphate. The modified mutant enzymes showed kcat values of 19.6-0.065% of that of the wild-type enzyme in the overall reaction, and were 10(6)-10(8) times more active than the K239C mutant enzyme. These results suggest that omega-amino groups of the introduced residues of the modified mutant enzyme serve as a catalytic base, and catalysis of the enzyme was affected by the length of the functional side-chain.

Aspartate Aminotransferases↗
PubMed1994

Reconsideration of the essential role of a histidine residue of L-2-halo acid dehalogenase.

His20 of L-2-halo acid dehalogenase from Pseudomonas cepacia MBA4 was suggested to serve as a catalytic base [Biochem. J. (1993) 292, 69-74]. In this study, we substituted Asn or Leu for His19 of L-2-halo acid dehalogenase from Pseudomonas sp. YL, which corresponds to His20 of the P. cepacia enzyme. Although the substrate specificity was affected by the substitution, the susceptibilities of substrate halo acids were not substantially diminished, and the Km and kcat values of the mutant enzymes for L-2-chloropropionate were not significantly different from those of the wild-type enzyme. In addition, the wild-type and mutant enzymes showed the same pH optimum. Accordingly, His19 is not essential for catalysis of L-2-halo acid dehalogenase.

Aspartic Acid↗
PubMed1994

Construction and characterization of chimeric enzyme consisting of an amino-terminal domain of phenylalanine dehydrogenase and a carboxy-terminal domain of leucine dehydrogenase.

Phenylalanine dehydrogenase of Thermoactinomyces intermedius acts preferentially on L-phenylalanine and L-tyrosine, whereas leucine dehydrogenase of Bacillus stearothermophilus acts almost exclusively on L-leucine and some other branched-chain L-amino acids. The two enzymes share a sequence similarity (47%). Aiming at elucidation of the mechanism of substrate recognition by the two amino acid dehydrogenases, we have genetically constructed a chimeric enzyme consisting of an N-terminal domain of phenylalanine dehydrogenase containing the substrate-binding region and a C-terminal domain of leucine dehydrogenase containing the NAD(+)-binding region. The chimeric enzyme purified to homogeneity acted on phenylalanine with a specific activity of 6% of that of the parental phenylalanine dehydrogenase and showed a broad substrate specificity in the oxidative deamination, like phenylalanine dehydrogenase. However, it acted much more effectively than phenylalanine dehydrogenase on isoleucine and valine. Its Km values for L-phenylalanine and L-leucine were similar to those of phenylalanine dehydrogenase. The substrate specificity of the chimeric enzyme in the reductive amination was an admixture of those of the two parent enzymes. These results suggest that the two domains of phenylalanine dehydrogenase and leucine dehydrogenase probably can fold independently. Accordingly, their chimera forms a new active enzyme which consists of their N- and C-terminal domains containing the substrate- and coenzyme-binding regions, respectively. However, the two domains of chimeric enzyme interact and communicate with each other to form a new active site and consistently show the new substrate specificity.

Amino Acid Oxidoreductases↗
PubMed1994

Identification of active site lysyl residues of phenylalanine dehydrogenase by chemical modification with methyl acetyl phosphate combined with site-directed mutagenesis.

A monoanionic acetylation reagent, methyl acetyl phosphate, was used to acetylate lysyl residues of the recombinant thermostable phenylalanine dehydrogenase from Thermoactinomyces intermedius. The enzyme was irreversibly inactivated with the reagent in a time- and dose-dependent manner. Simultaneous addition of substrate and coenzyme markedly protected the enzyme from inactivation. Acetylated lysyl residues presumably occurring at the active site were determined by differential modification; the enzyme was first modified with a cold reagent in the presence of both substrate and coenzyme and, after removal of the added substances by gel filtration, was then labeled with a radioactive reagent. At least 7 lysyl residues per enzyme subunit were radiolabeled by this method. To further specify the lysyl residue(s) whose modification results in inactivation of the enzyme, 5 lysyl residues highly conserved in various amino acid dehydrogenase sequences were replaced with Ala by site-directed mutagenesis. Although all of the single mutant enzymes were inactivated with the reagent as effectively as the wild-type enzyme, a double mutant enzyme in which both Lys-69 and Lys-81 were replaced with Ala was found to be inactivated very slowly. These results suggest that the reagent can acetylate both of these lysyl residues and inactivate the enzyme. Kinetic analyses of the single Lys-69 and Lys-81 mutant enzymes revealed that they are involved in substrate binding and catalysis, respectively, like the corresponding residues in the homologous leucine dehydrogenase.

Amino Acid Oxidoreductases↗
PubMed1994

Characterization of mice bearing subclones of colon 26 adenocarcinoma disqualifies interleukin-6 as the sole inducer of cachexia.

A subclone (clone 20) of chemically induced, murine colon adenocarcinoma with a potent ability to induce cachexia and another subclone (clone 5) without such an activity were transplanted to syngeneic mice (CDF1) and their tissue weights, blood components and cytokine levels in sera were compared. Mice transplanted with clone 20 showed a profound body-weight loss by 15 days after inoculation when the tumor accounted for less than 1% of the body weight, along with marked reduction of food and water intakes. Thereafter, they transiently gained in body weight with restoration of food and water intakes. Thus, the change in body weight was biphasic and not proportional to the tumor size. Body fat was lost preferentially, accompanied with a decrease in plasma triglyceride levels. The thymus contracted remarkably, and the peripheral lymphocyte count decreased extensively. Mice transplanted with clone 5, in contrast, did not show any of these changes characteristic of cachexia. Serum concentration of interleukin-6, which has been proposed as the principal inducer of cachexia in mice with colon 26, increased in mice with clone 5 to levels comparable to those in mice with clone 20. The changes in mice bearing clone 20 could not all be explained in terms of known biological activities of interleukin-6. Additional unknown factors, therefore, are presumed to contribute to cachexia in mice with clone 20. Identification of them should be helpful in the care of cachectic patients.

Adenocarcinoma↗
PubMed1994

Purification and characterization of thermostable and nonthermostable 2-haloacid dehalogenases with different stereospecificities from Pseudomonas sp. strain YL.

Two novel hydrolytic dehalogenases, thermostable L-2-haloacid dehalogenase (L-DEX) inducibly synthesized by 2-chloropropionate (2-CPA) and nonthermostable DL-2-haloacid dehalogenase (DL-DEX) induced by 2-chloroacrylate, were purified to homogeneity from Pseudomonas sp. strain YL. DL-DEX consisted of a monomer with a molecular weight of about 36,000 and catalyzed the dehalogenation of L and D isomers of 2-CPA to produce D- and L-lactates, respectively. It acted on 2-haloalkanoic acids with a carbon chain length of 2 to 4. The maximum activity on DL-2-CPA was found at pH 10.5 and 45 degrees C. L-DEX, composed of two subunits with identical molecular weights of 27,000, catalyzes the dehalogenation of L-2-haloalkanoic acids to produce the corresponding D-2-hydroxyalkanoic acids. The enzyme acts not only on short-carbon-chain 2-haloacids such as monochloroacetate and monoiodoacetate in aqueous solution but also on long-carbon-chain 2-haloacids such as 2-bromohexadecanoate in n-heptane. L-DEX is thermostable: it retained its full activity upon heating at 60 degrees C for 30 min. The pH and temperature optima for dehalogenation of L-2-CPA were 9.5 and 65 degrees C, respectively. L-DEX was strongly inhibited by modification of carboxyl groups with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and Woodward reagent K, but DL-DEX was not.

Amino Acid Sequence↗
PubMed1994

Comparative studies of genes encoding thermostable L-2-halo acid dehalogenase from Pseudomonas sp. strain YL, other dehalogenases, and two related hypothetical proteins from Escherichia coli.

We have determined the nucleotide sequence of the gene encoding thermostable L-2-halo acid dehalogenase (L-DEX) from the 2-chloroacrylate-utilizable bacterium Pseudomonas sp. strain YL. The open reading frame consists of 696 nucleotides corresponding to 232 amino acid residues. The protein molecular weight was estimated to be 26,179, which was in good agreement with the subunit molecular weight of the enzyme. The gene was efficiently expressed in the recombinant Escherichia coli cells: the amount of L-DEX corresponds to about 49% of the total soluble proteins. The predicted amino acid sequence showed a high level of similarity to those of L-DEXs from other bacterial strains and haloacetate dehalogenase H-2 from Moraxella sp. strain B (38 to 57% identity) but a very low level of similarity to those of haloacetate dehalogenase H-1 from Moraxella sp. strain B (10%) and haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 (12%). By searching the protein amino acid sequence database, we found two E. coli hypothetical proteins similar to the Pseudomonas sp. strain YL L-DEX (21 to 22%).

Amino Acid Sequence↗
PubMed1993

Expression of glr (murI, dga) gene encoding glutamate racemase in Escherichia coli.

The murI (dga) gene of Escherichia coli is required for the biosynthesis of D-glutamate, an essential component of bacterial peptidoglycan (Doublet, P., van Heijetnoort, J., and Mengin-Lecreulx, D. (1992) J. Bacteriol. 174, 5772-5779; Dougherty, T. J., Thanassi, J. A., and Pucci, M. J. (1993) J. Bacteriol. 175, 111-116), but its gene product has not been identified. We found that the amino acid sequence of protein deduced from the nucleotide sequence of the open reading frame of murI gene (ORF1) shows a significant homology with that of glutamate racemase of Pediococcus pentosaceus. The amino acid sequence of glutamate racemase of Lactobacillus fermenti recently reported also shows a homology with the deduced amino acid sequence of ORFI (Gallo, K. A., and Knowles, J. R. (1993) Biochemistry 32, 3981-3990). The murI (dga) gene was ligated into a plasmid, pKK223-3, with a designed ribosome binding site and expressed in E. coli JM109 cells. Glutamate racemase was produced by the transformant cells, whereas the enzyme was not found in the host cells. Accordingly, we newly termed the gene glr, which is more relevant than murI and dga. We partially purified the enzyme to characterize it. The enzyme consists of two identical subunits with a molecular weight of about 31,000 in contrast to the P. pentosaceus enzyme, a monomer protein.

Amino Acid Isomerases↗