N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

Properties of Crystalline ω-Amino Acid : Pyruvate Aminotransferase of Pseudomonas sp. F–126

ω-Amino acid: pyruvate aminotransferase, purified to homogeneity and crystallized from a Pseudomonas sp. F–126, has a molecular weight of 172,000 or 167,000±3000 as determined by the gel-filtration or sedimentation equilibrium method, respectively. The enzyme catalyzes the transamination between various ω-amino acids or amines and pyruvate which is the exclusive amino acceptor. α-Amino acids except l-α-alanine are inert as amino donor. The Michaelis constants are 3.3 mm for β-alanine, 19 mm for 2-aminoethane sulfonate and 3.3 mm for pyruvate. The enzyme has a maximum activity in the pH range of 8.5~10.5. The enzyme is stable at pH 8.0~10.0 and at up to 65°C at pH 8.0. Carbonyl reagents strongly inhibit the enzyme activity. Pyridoxal 5′-phosphate and pyridoxamine 5′-phosphate reactivate the enzyme inactivated by carbonyl reagents. The inhibition constants were determined to be 0.73 mm for d-penicillamine and 0.58 mm for d-cycloserine. Thiol reagents, chelating agents and l-α-amino acids showed no effect on the enzyme activity.     

Functional Characterization of D-Galacturonic Acid Reductase,a Key Enzyme of the Ascorbate Biosynthesis Pathway,from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38–39 kDa, as judged by SDS–PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5±4.5 μM and uronic acids, such as D-galacturonic acid (Km=3.79±0.5 mM) and D-glucuronic acid (Km=4.67±0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP⁺. The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H₂O₂, suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Biosynthetic Threonine Deaminase from Proteus morganii

Biosynthetic threonine deaminase was purified to an apparent homogeneous state from the cell extract of Proteus morganii, with an overall yield of 7.5%. The enzyme had a s⁰_20,w of 10.0 S, and the molecular weight was calculated to be approximately, 228,000. The molecular weight of a subunit of the enzyme was estimated to be 58,000 by sodium dodecyl sulfate gel electrophoresis. The enzyme seemed to have a tetrameric structure consisting of identical subunits. The enzyme had a marked yellow color with an absorption maximum at 415 nm and contained 2 mol of pyridoxal 5′-phosphate per mol. The threonine deaminase catalyzed the deamination of l-threonine, l-serine, l-cysteine and β-chloro-l-alanine. Km values for l-threonine and l-serine were 3.2 and 7.1 mm, respectively. The enzyme was not activated by AMP, ADP and ATP, but was inhibited by l-isoleucine. The Ki for l-isoleucine was 1.17 mm, and the inhibition was not recovered by l-valine. Treatment with mercuric chloride effectively protected the enzyme from inhibition by l-isoleucine.     

New Nematicidal Metabolites from a Fungus,Irpex lacteus

N-Benzoyl-l-alanine amidohydrolase was purified from a cell-free extract of Corynebacterium equi H-7 which was grown in a medium containing hippuric acid as the sole carbon source. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. The molecular weight was 230,000 and the enzyme consisted of six subunits, identical in molecular weight (approximately 40,000). The isoelectric point of the enzyme was pH 4.6. The optimum pH of the enzyme reaction was 8.0 and the enzyme was stable from pH 7.0 to 8.0. The enzyme hydrolyzed N-benzoyl-l-alanine, N-benzoylglycine, and N-benzoyl-l-aminobutyric acid. The Km values for these substrates were 4.3 mm, 6.7 mm, and 4.3 mm, respectively. The enzyme was activated by Co²⁺.     

d-Gluconate Dehydrogenase, 2-Keto-d-gluconate Yielding,from Gluconobacter dioxyacetonicus: Purification and Characterization

d-Gluconate dehydrogenase catalyzing the oxidation of d-gluconate to 2-keto-d-gluconate was solubilized with Triton X-100 from the membrane of Gluconobacter dioxyacetonicus IFO 3271 and purified to an almost homogeneous state by chromatographies on DEAE-cellulose and CM-Toyopearl in the presence of 0.1% Triton X-100. The enzyme had three subunits with molecular weights of 64,000, 45,000 and 21,000, and contained approximately 2 mol of heme per mol of the enzyme. The prosthetic group of the dehydrogenase was found to be a flavin covalently bound to the enzyme protein. The substrate specificity of the purified enzyme was very strict for d-gluconate and the apparent Michaelis constant for d-gluconate was 2.2 mm. The optimum pH and temperature of the purified enzyme were 6.0 and 40°C, respectively.     

Branched Chain Amino Acid Aminotransferase of Pseudomonas sp

Branched chain amino acid aminotransferase was partially purified from Pseudomonas sp. by ammonium sulfate fractionation, aminohexyl-agarose and Bio-Gel A-0.5 m column chromatography.

This enzyme showed different substrate specificity from those of other origins, namely lower reactivity for l-isoleucine and higher reactivity for l-methionine.

Km values at pH 8.0 were calculated to be 0.3 mm for l-leucine, 0.3 mm for α-ketoglutarate, 1.1 mm for α-ketoisocaproate and 3.2 mm for l-glutamate.

This enzyme was activated with β-mercaptoethanol, and this activated enzyme had different kinetic properties from unactivated enzyme, namely, Km values at pH 8.0 were calculated to be 1.2 mm for l-leucine, 0.3 mm for α-ketoglutarate.

Isocaproic acid which is the substrate analog of l-leucine was competitive inhibitor for pyridoxal form of unactivated and activated enzymes, and inhibitor constants were estimated to be 6 mm and 14 mm, respectively.     

Purification and Characterization of Novel N-Acyl-D-aspartate Amidohydrolase from Alcaligenes xylosoxydans subsp. xylosoxydans A-6

Alcaligenes xylosoxydans subsp. xylosoxydans A-6 (Alcaligenes A-6) produced N-acyl-D-aspartate amidohydrolase (D-AAase) in the presence of N-acetyl-D-aspartate as an inducer. The enzyme was purified to homogeneity. The enzyme had a molecular mass of 56 kDa and was shown by sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) to be a monomer. The isoelectric point was 4.8. The enzyme had maximal activity at pH 7.5 to 8.0 and 50°C, and was stable at pH 8.0 and up to 45°C. N-Formyl (K_m=12.5 mM), N-acetyl (K_m=2.52 mM), N-propionyl (K_m=0.194 mM), N-butyryl (K_m=0.033 mM), and N-glycyl (K_m =1.11 mM) derivatives of D-aspartate were hydrolyzed, but N-carbobenzoyl-D-aspartate, N-acetyl-L-aspartate, and N-acetyl-D-glutamate were not substrates. The enzyme was inhibited by both divalent cations (Hg²⁺, Ni²⁺, Cu²⁺) and thiol reagents (N-ethylmaleimide, iodoacetic acid, dithiothreitol, and p-chloromercuribenzoic acid). The N-terminal amino acid sequence and amino acid composition were analyzed.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

Purification,Characterization, and Molecular Cloning of a Pyranose Oxidase from the Fruit Body of the Basidiomycete,Tricholoma matsutake

A new H₂O₂-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H₂O₂ and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V _max, K _m, and k _cat for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50°C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Phenol Ethers and Terpenes of Six Heterotropa Species Distributed in the Ryukyu Islands and Formosa

A new intracellular peptidase, which we call “d-peptidase S,” was purified from Nocardia orientalis IFO 12806 (ISP 5040). The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight and the isoelectric point were estimated to be 52,000 and 4.9, respectively. The optimum pH for the hydrolysis of d-leucyl-d-leucine was 8.0 to 8.1, and the optimum temperature was 36°C. The purified enzyme usually hydrolyzed the peptide bonds preceding the hydrophobic D-amino acids of dipeptides. Tri- and tetra-peptides extending to the amino terminus of such peptides were also hydrolyzed. Therefore, the enzyme is a carboxylpeptidase-like peptidase specific to d-amino acid peptides. The Km values for d-leucyl-d-leucine and l-leucyl-d-leucine were 0.21 × 10^-3 and 0.44 × 10^-3 m respectively. The activity was inhibited by several sulfhydryl reagents and two chelators, 8-hydroxyquinoline and o-phenanthroline.     

Studies on the Polymorphism of l-Glutamic Acid

The effects on the polymorphic crystallization of l-glutamic acid were examined of many substances including amino acids, inorganic salts, surface active agents, and sodium salt or hydrochloride of l-glutamic acid, when contained in the mother liquor.

The co-existence of amino acids, especially of l-aspartic acid, l-phenylalanine, l-tyrosine, l-lcucine and l-cystine contributed to the crystallization of l-glutamic acid in α-form, and these amino acid showed an inhibitory action on the transition of α-crystals as the solid phase in the aqueous solution, to β-crystals.

In the presence of a large amount of l-glutamate or the hydrochloride at the time of nucleation of l-glutamic acid, mostly β-crystals appeared even in the presence of the amino acids named above.     

Distribution of 7-Keto-8-aminopelargonic Acid Synthetase in Bacteria and the Control Mechanism of the Enzyme Activity

The 7-keto-8-aminopelargonic acid (KAPA) synthetase activities of cell-free extracts from various bacteria were investigated. The experiments on the substrate specificity of KAPA synthetase, using crude cell-free extracts from bacteria having high enzyme activity, showed that l-serine and pyruvic acid could replace l-alanine, but that, when the enzyme was partially purified, these compounds were not effective. Many kinds of amino acids such as l-cysteine, l-serine, d-alanine, glycine, d-histidine, and l-histidine, inhibited the enzyme activity. This inhibition was found to be competitive with l-alanine. Pyridoxal 5′-phosphate, which is a cofactor of the enzyme, also inhibited the enzyme activity at high concentrations. The repression of KAPA synthetase by biotin occurred in Bacillus subtilis and B. sphaericus but not in Micrococcus roseus and Pseudomonas fluorescens, even at a concentration of 1000 mµg per ml of biotin.     

Production of Xylanase by Streptomyces sp., Using Non-metabolizable Inducer

meso-Diaminopimelate dehydrogenase (EC 1.4.1.16) was purified to homogeneity from Corynebacterium glutamicum ATCC 13032. The enzyme had a molecular weight of about 70,000 and consisted of two subunits identical in molecular weight. The enzyme was highly specific for meso-2,6-diaminopimelate. The pH optima for deamination and amination were about 9.8 and 7.9, respectively. The Michaelis constants were 3.1mm for meso-2,6-diaminopimelate, 0.12mm for NADP⁺, 0.28 mm for l-2-amino-6-ketopimelate, 36 mm for ammonia, and 0.13 mm for NADPH. d and l isomers of 2,6-diaminopimelate competitively inhibited the oxidative deamination of meso-2,6-diaminopimelate. The enzyme was distributed in a wider range of bacterial species than reported previously [Misono et al., J. Bacteriol., 137, 22 (1979)] when assayed by a sensitive formazan formation method.     

DAHP Synthetase and Its Control in Corynebacterium glutamicum

Properties of 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthetase from Corynebacterium glutamicum were examined using the cell free extract. The optimum pH for the reaction was broad ranging from 5.5 to 7.0 and the optimum temperature was 37°C. Co²⁺ inhibited the enzyme activity at 20°C, whereas Co²⁺ apparently stimulated the enzyme activity at 37°C because the ion protected the enzyme from inactivation at 37°C. Co²⁺ reversed the inhibition of the enzyme activity by EDTA. The activity of DAHP synthetase was feedback inhibited only weakly by l-phenylalanine, l-tyrosine or l-tryptophan alone, but was strongly inhibited synergistically by l-phenylalanine and l-tyrosine. l-Tryptophan enhanced the inhibition by the pair of l-tyrosine and l-phenylalanine. Maximal inhibition was near 90 % in the simultaneous presence of the three amino acids. Sensitivity of the enzyme to the inhibitors was lost during the purification process of the enzyme or during the reaction at 37°C. Especially sensitivity to l-tryptophan was easily lost. Co²⁺ protected the enzyme from the desensitization. Mutants resistant to p-fluorophenylalanine plus l-tyrosine (or 3-aminotyrosine) had DAHP synthetase which was released from the feedback inhibition by the three amino acids. The formation of the enzyme was not affected by aromatic amino acids.     

Substrate Specificity of Alkaline Proteinases from Aspergillus sydowi and Aspergillus sulphureus

l-Fucose (l-galactose) dehydrogenase was isolated to homogeneity from a cell-free extract of Pseudomonas sp. No 1143 and purified about 380-fold with a yield of 23 %. The purification procedures were: treatment with polyethyleneimine, ammonium sulfate fractionation, chromatographies on phenyl-Sepharose and DEAE-Sephadex, preparative polyacrylamide gel electrophoresis, and gel filtration on Sephadex G-100. The enzyme had a molecular weight of about 34,000. The optimum pH was at 9 — 10.5 and the isoelectric point was at pH 5.1. l-Fucose and l-galactose were effective substrates for the enzyme reaction, but d-arabinose was not so much. The anomeric requirement of the enzyme to l-fucose was the β-pyranose form, and the reaction product from l-fucose was l-fucono- lactone. The hydrogen acceptor for the enzyme reaction wasNADP⁺, and NAD ⁺ could be substituted for it to a very small degree. Km values were 1.9mm, 19mm, 0.016mm, and 5.6mm for l-fucose, l- galactose, NADP⁺, and NAD⁺, respectively. The enzyme activity was strongly inhibited by Hg^{2 +}, Cd^{2 +}, and PCMB, but metal-chelating reagents had almost no effect. In a preliminary experiment, it was indicated that the enzyme may be usable for the measurement of l-fucose.     

Inhibition of l-Alanine Adding Enzyme by Glycine

l-Alanine adding enzymes from Bacillus subtilis and Bacillus cereus which catalyzed l-alanine incorporation into UDPMurNAc were partially purified and the properties of the enzymes were examined. The enzyme from B. subtilis was markedly stimulated by reducing agents including 2-mercaptoethanol, dithiothreitol, glutathione and cysteine. Mn²⁺ and Mg²⁺ activated l-alanine adding activity and their optimal concentrations were 2 to 5 mm and 10 mm, respectively. The optimum pH was 9.5 and the Km for l-alanine was 1.8×10^?4m. l-Alanine adding reaction was strongly inhibited by p-chloromercuribenzoate and N-ethyl-maleimide. Among glycine, l- and d-amino acids and glycine derivatives, glycine was the most effective inhibitor of the l-alanine adding reaction. The enzyme from B. cereus was more resistant to glycine than that from B. subtilis. Glycine was incorporated into UDPMurNAc in place of l-alanine, and the Ki for glycine was 4.2×l0^?3m with the enzyme from B. subtilis. From these data, the growth inhibition of bacteria by glycine is discussed.     

Studies on Metabolism of Pyrrolidone Carboxylic Acid in Bacteria

l-Glutamic acid was formed from d-, l-, and dl-PCA with cell-free extract of Pseudomonas alcaligenes ATCC-12815 grown in the medium containing dl-PCA as a sole source of carbon and nitrogen. The enzyme(s) involved in this conversion reaction was distributed in the soluble fraction within the cell and in 0.5 saturated fraction at the fractionation procedure with the saturation of ammonium sulfate. Optimum pH of this enzyme(s) lied at pH 8.5 and optimum temperature was 30°C. Cu (5 × 10^?3 m) inhibited the reaction considerably while Ca or Fe accelerated it. PALP (1×10^?3 m) also gave an enhanced activity to some extent. The enzyme preparation converted dextro-rotatory enan-thiomorph of PCA to its laevo-rotatory one which in turn was not converted to the opposite rotation direction by this enzyme. Furthermore, the preparation did not, if any, show d-glutamic acid racemase activity. Isotopic experiments with using dl-PCA-1-¹⁴C revealed that l-glutamic acid-1-¹⁴C was formed by the cleavage of –CO–NH– bond of pyrrolidone ring of PCA. It was concluded that dl-PCA when assimilated by the present bacterium is at first transformed to l-PCA by the optically isomerizing enzyme and subsequently is cleaved to l-glutamic acid probably by the PCA hydrolysing enzyme.     

Gene Cloning of α-Methylserine Aldolase from Variovorax paradoxus and Purification and Characterization of the Recombinant Enzyme

The α-methylserine aldolase gene from Variovorax paradoxus strains AJ110406, NBRC15149, and NBRC15150 was cloned and expressed in Escherichia coli. Formaldehyde release activity from α-methyl-L-serine was detected in the cell-free extract of E.coli expressing the gene from three strains. The recombinant enzyme from V. paradoxus NBRC15150 was purified. The V _max and K _m of the enzyme for the formaldehyde release reaction from α-methyl-L-serine were 1.89 μmol min^?1 mg^?1 and 1.2 mM respectively. The enzyme was also capable of catalyzing the synthesis of α-methyl-L-serine and α-ethyl-L-serine from L-alanine and L-2-aminobutyric acid respectively, accompanied by hydroxymethyl transfer from formaldehyde. The purified enzyme also catalyzed alanine racemization. It contained 1 mole of pyridoxal 5′-phosphate per mol of the enzyme subunit, and exhibited a specific spectral peak at 429 nm. With L-alanine and L-2-aminobutyric acid as substrates, the specific peak, assumed to be a result of the formation of a quinonoid intermediate, increased at 498 nm and 500 nm respectively.     

Separation of l-Leucine-pyruvate and l-Leucine-α-ketoglutarate Transaminases in Acetobacter suboxydans and Identification of Their Reaction Products

l-Leucine-pyruvate and l-leucine-α-ketoglutarate(α-KGA) transaminases were separated by DEAE-cellulose column chromatography and partially purified to 200- and 50-fold, respectively, from the cell-free extract of Acetobacter suboxydans (Gluconobacter suboxydans IFO 3172). The optimum pH range of the former was 5.0~5.5 and that of the latter was 8.5~9.0. l-Leucine, l-citrulline, and l-methionine were the most effective amino donors for the l-leucine-pyruvate transaminase. Basic amino acids as well as aromatic amino acids were able to be amino donors for the transamination with pyruvate. α-KGA was effective as an amino acceptor for this enzyme. The l-leucine-α-KGA transaminase had the typical properties of the branched-chain amino acid transaminase in its substrate specificity.

The reaction products of the transaminations were identified. l-Alanine was formed from pyruvate and l-glutamate from α-KGA. α-Keto acids formed from various amino acids by the l-leucine-pyruvate transaminase were also identified.