Toshiharu Yagi

[17] Glutamate-aspartate transaminase from microorganisms

Publisher Summary Various methods have been developed for the assay of aspartate aminotransferase. Most of these depend on the determination of oxalacetate or L-glutamate. Out of these, the Karmen method is used most widely, particularly in the field of clinical analysis, in which oxalacetate is determined with malate dehydrogenase and NADH by following a decrease in absorbance at 340 nm. A new method introduced recently, determines 2-oxoglutarate produced in the reverse reaction is with 2-hydroxyglutarate dehydrogenase. A method for the determination of aspartate aminotransferase activity after electrophoretic separation of the isoenzymes has also been developed. The purification procedure consists of several steps: preparation of cell-free extract, ammonium sulfate fractionation, first Diethylaminoethyl (DEAE)-cellulose column chromatography, heat treatment, Sephacryl S-200 chromatography, second DEAE-cellulose chromatography, and hydroxyapatite chromatography.

Poly-γ-glutamate depolymerase of Bacillus subtilis : production, simple purification and substrate selectivity

Abstract The Bacillus subtilis pgdS gene, which is located at the immediate downstream of the pgs operon for poly-γ-glutamate (PGA) biosynthesis, encodes a PGA depolymerase. The pgdS gene product shows the structural feature of a membrane-associated protein. The mature form of the gene product, identified as a B. subtilis extracellular protein, was produced in Escherichia coli clone cells. Since the mature PGA depolymerase has been modified with the histidine-tag at its C-terminus, it could be simply purified by metal-chelating affinity chromatography. This purified enzyme digested PGAs from B. subtilis ( d -glutamate content, 70%) and from Bacillus megaterium (30%) in an endopeptidase-like fashion. In contrast, PGA from Natrialba aegyptiaca, which consists only of l -glutamate, was resistant to the enzyme, suggesting that, unlike fungal PGA endo-depolymerases, the bacterial enzyme recognizes the d -glutamate unit in PGA.

Purification, molecular cloning, and catalytic activity of Schizosaccharomyces pombe pyridoxal reductase: A possible additional family in the aldo-keto reductase superfamily

Pyridoxal reductase (PL reductase), which catalyzes reduction of PL by NADPH to form pyridoxine and NADP+, was purified from Schizosaccharomyces pombe. The purified enzyme was very unstable but was stabilized by low concentrations of various detergents such as Tween 40. The enzyme was a monomeric protein with the native molecular weight of 41,000 ± 1,600. The enzyme showed a single absorption peak at 280 nm (E 1% = 10.0). PL and 2-nitrobenzaldehyde were excellent substrates, and no measurable activity was observed with short chain aliphatic aldehydes; substrate specificity of PL reductase was obviously different from those of yeast aldo-keto reductases (AKRs) so far purified. The peptide sequences of PL reductase were identical with those in a hypothetical 333-amino acid protein from S. pombe (the DDBJ/EMBL/GenBankTM accession numberD89205). The gene corresponding to this protein was expressed inEscherichia coli, and the purified protein was found to have PL reductase activity. The recombinant PL reductase showed the same properties as those of native PL reductase. PL reductase showed only low sequence identities with members of AKR superfamily established to date; it shows the highest identity (18.5%) with humanShaker-related voltage-gated K+ channel β2 subunit. The elements of secondary structure of PL reductase, however, distributed similarly to those demonstrated in the three-dimensional structure of human aldose reductase except that loop A region is lost, and loop B region is extended. Amino acid residues involved in substrate binding or catalysis are also conserved. Conservation of these features, together with the major modifications, establish PL reductase as the first member of a new AKR family, AKR8.

Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp

An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme.

PURIFICATION AND PROPERTIES OF BACILLUS COAGULANS CYCLOMALTODEXTRIN GLUCANOTRANSFERASE

Abstract Cyclomaltodextrin glucanotransferase (CGTase), produced in a culture filtrate by Bacillus coagulans , was purified to a single, homogeneous protein. It has a monomeric structure with a molecular weight of 65,000, isoelectric point of 4.6, and contains 2 mol of Ca 2+ per mol of the enzyme. The enzyme was most active at pH 6.0 and at 70°C. It did not lose its activity by heat treatment at 70°C for 10 min in the presence of CaCl 2 in the pH range of 5.5∼9.5, and by incubation in the pH range of 5.0∼10.5 at 4°C for one month. The enzyme converted about 60% of potato starch to cyclodextrins for 20 h at 50°C, and the ratio of α-: β-: γ-cyclodextrin produced was 8.1:8.9:1.0 B. coagulans CGTase was compared with B. macerans CGTase which was purified by the same method.

Purification, Molecular Cloning, and Characterization of Pyridoxine 4-Oxidase from Microbacterium luteolum

Pyridoxine 4-oxidase (EC 1.1.3.12, PN 4-oxidase), which catalyzes the oxidation of PN by oxygen or other hydrogen acceptors to form pyridoxal and hydrogen peroxide or reduced forms of the acceptors, respectively, was purified for the first time to homogeneity from Microbacterium luteolum YK-1 (=Aureobacterium luteolum YK-1).1 The purified enzyme required FAD for its catalytic activity and stability. The enzyme was a monomeric protein with the subunit molecular mass of 53,000±1,000 Da. PN was the only substrate as the hydrogen donor. Oxygen, 2,6-dichloroindophenol, and vitamin K3 were good substrates as the hydrogen acceptor. The gene (pno) encoding PN 4-oxidase was cloned. The gene encodes a protein of 507 amino acid residues corresponding to the molecular mass of the subunit. PN 4-oxidase was expressed in Escherichia coli and found to have the same properties as the native enzyme from M. luteolum YK-1. Comparisons of primary and secondary structures with other proteins showed that the enzyme belongs to the GMC oxidoreductase family. M. luteolum YK-1 has four plasmids. The pno gene was found on a chromosomal DNA. Search for genes similar in sequence in other organisms suggested that a nitrogen-fixing symbiotic bacterium, Mesorhizobium loti, which harbors two plasmids, has a PN degradation pathway I in chromosomal DNA.

Molecular cloning, expression, and properties of an unusual aldo-keto reductase family enzyme, pyridoxal 4-dehydrogenase, that catalyzes irreversible oxidation of pyridoxal.

Microbacterium luteolum YK-1 has pyridoxine degradation pathway I. We have cloned the structural gene for the second step enzyme, pyridoxal 4-dehydrogenase. The gene consists of 1,026-bp nucleotides and encodes 342 amino acids. The enzyme was overexpressed under cold shock conditions with a coexpression system and chaperonin GroEL/ES. The recombinant enzyme showed the same properties as the M. luteolum enzyme. The primary sequence of the enzyme was 54% identical with that of d-threo-aldose 1-dehydrogenase from Agrobacterium tumefaciens, a probable aldo-keto reductase (AKR). Upon multiple alignment with enzymes belonging to the 14 AKR families so far reported, pyridoxal 4-dehydrogenase was found to form a new AKR superfamily (AKR15) together with A. tumefaciens d-threo-aldose 1-dehydrogenase and Pseudomonas sp. l-fucose dehydrogenase. These enzymes belong to a distinct branch from the two main ones found in the phylogenic tree of AKR proteins. The enzymes on the new branch are characterized by their inability to reduce the corresponding lactones, which are produced from pyridoxal or sugars. Furthermore, pyridoxal 4-dehydrogenase prefers NAD+ to NADP+ as a cofactor, although AKRs generally show higher affinities for the latter.

Crystal Structure of Pyridoxamine-Pyruvate Aminotransferase from Mesorhizobium loti MAFF303099

Pyridoxamine-pyruvate aminotransferase (PPAT; EC 2.6.1.30) is a pyridoxal 5′-phosphate-independent aminotransferase and catalyzes reversible transamination between pyridoxamine and pyruvate to form pyridoxal and l-alanine. The crystal structure of PPAT from Mesorhizobium loti has been solved in space group P43212 and was refined to an R factor of 15.6% (Rfree = 20.6%) at 2.0Å resolution. In addition, the structures of PPAT in complexes with pyridoxamine, pyridoxal, and pyridoxyl-l-alanine have been refined to R factors of 15.6, 15.4, and 14.5% (Rfree = 18.6, 18.1, and 18.4%) at 1.7, 1.7, and 2.0Å resolution, respectively. PPAT is a homotetramer and each subunit is composed of a large N-terminal domain, consisting of seven β-sheets and eight α-helices, and a smaller C-terminal domain, consisting of three β-sheets and four α-helices. The substrate pyridoxal is bound through an aldimine linkage to Lys-197 in the active site. The α-carboxylate group of the substrate amino/keto acid is hydrogen-bonded to Arg-336 and Arg-345. The structures revealed that the bulky side chain of Glu-68 interfered with the binding of the phosphate moiety of pyridoxal 5′-phosphate and made PPAT specific to pyridoxal. The reaction mechanism of the enzyme is discussed based on the structures and kinetics results.

Purification and characterization of pyridoxal 4-dehydrogenase from Aureobacterium luteolum.

A pyridoxal dehydrogenase was purified to homogeneity from Aureobacterium luteolum, which can use pyridoxine as a carbon and nitrogen source, and characterized. The enzyme was a dimeric protein with a subunit molecular weight of 38,000. It had several properties distinct from those of the partially purified enzyme from Pseudomonas MA-1. The optimum pH (8.0–8.5) was 0.8–1.3 lower than that of the Pseudomonas enzyme. The Aureobacterium enzyme showed much higher and lower affinities for NAD+ (Km, 0.140±0.008 mM) and pyridoxal (0.473±0.109 mM), respectively, than those of the Pseudomonas enzyme. The Aureobacterium enzyme could use NADP+ as a substrate: the reactivity was 6.5% of NAD+. The enzyme was much more tolerant to metal-chelating agents. Irreversibility of the enzymatic reaction was shared by the two enzymes. No aldehyde dehydrogenase showed similarity to the amino-terminal amino acid sequence of the enzyme.

Determination of human aspartate aminotransferase isoenzymes by their differential sensitivity to proteases

The effect of various proteases (trypsin, chymotrypsin, subtilisin, protease 401, and thermolysin) on the mitochondrial isoenzyme (m-AST) and cytoplasmic isoenzyme (c-AST) of human and swine aspartate aminotransferase (AST;EC 2.6.1.1) was evaluated. All procedures including the reaction with proteases and the subsequent determination of the AST activity were carried out in an automatic analyzer. The mammalian c-AST was efficiently inactivated by chymotrypsin, subtilisin and protease 401 while m-AST activity decreased very slowly with these proteases. Thermolysin and trypsin showed much less effect on c-AST activity. Especially, chymotrypsin at concentrations of 0.5-1.0 g/L inactivated human c-AST almost completely but showed no detectable inactivating effect on m-AST. Thus chymotrypsin appears to be the most suitable protease for the differential determination of AST isoenzymes in human serum. Further studies on the effects of proteases with AST from other species showed that Escherichia coli AST resembled mammalian m-AST while Pseudomonas AST resembled c-AST.