Katsuyuki Tanizawa

Coenzyme B12-dependent diol dehydrase: Purification, subunit heterogeneity, and reversible association

Abstract A purification procedure for diol dehydrase ( dl -1,2-propanediol hydro-lyase, EC 4.2.1.28) of Klebsiella pneumoniae (Aerobacter aerogenes) ATCC 8724 has been developed which gives the highest specific activity for this enzyme obtained so far. The purified enzyme is homogeneous by the criteria of ultracentrifugation ( s 20,w = 8.9 S) and disc gel electrophoresis in the presence of substrate. The molecular weight of approximately 230,000 was obtained by gel filtration and ultracentrifugal sedimentation equilibrium. The enzyme is composed of components F and S whose molecular weights were determined to be approximately 26,000 and 200,000, respectively, by gel filtration. The incubation of both components F and S with the substrate leads to complete reassociation of the components. Disc gel electrophoresis in the presence of sodium dodecyl sulfate and terminal amino acid analyses indicate that component S consists of at least four nonidentical subunits. The reversible association and heterogeneity of the subunits were also demonstrated with the crude enzyme by immunoelectrophoresis.

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

Abstract We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.

Conversion of α-keto acids to D-amino acids by coupling of four enzyme reactions

Abstract We developed a new procedure for stereospecific conversion of various α-keto acids to the corresponding d -amino acids with four thermostable enzymes: d -amino acid aminotransferase, alanine racemase, l -alanine dehydrogenase and formate dehydrogenase. Optically pure d -enantiomers of glutamate, phenylalanine and tyrosine were obtained with high conversion rates.

Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus

Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis. The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically). The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees. The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell. There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

A spectrophotometric method for the determination of aminopeptidase activity with leucine dehydrogenase

L-Leucine dehydrogenase purified from Bacillus megaterium and Bacillus sphearicus was used for the determination of serum aminopeptidase activity with L-leucinamide as a substrate. L-Leucine produced by aminopeptidase was determined by measurement of the increase in absorbance at 340 nm caused by the formation of NADH. This method is useful for the kinetic studies of the aminopeptidase and the enzyme assay of a large number of samples. The serum aminopeptidase can be characterized to give some valuable information in clinical diagnosis by comparison of the results obtained by the present method with those by the conventional method with L-leucyl-p-nitroanilide as a substrate.

Production of a novel tryptophan analog, β-1-indazole-L-alanine with tryptophan synthase of Escherichia coli

The tryptophan synthase α2β2 complex from Escherichia coli has been found to catalyze the β‐replacement reaction of L‐serine with indazole, an indole analog which has a nitrogen atom at the 2‐position (pyrazole ring). The reaction product was isolated and identified as β‐indazolealanine by mass spectrometric, elemental and NMR analyses. Careful assignment of 1H‐ and 13C‐signals with several NMR techniques revealed that the β‐carbon of the product alanine moiety was bound to the 1‐N‐position of the indazole ring. This is the first example of the β‐replacement reaction catalyzed by tryptophan synthase occurring at any other position than the 3‐position of indole analogs.

Purification and Characterization of Amino Acid Racemase with Very Broad Substrate Specificity from Aeromonas caviae

A bacterium which grows in a medium containing d-serine as a sole nitrogen source has been isolated from soil and identified as Aeromonas caviae. The bacterium had a high enzyme activity catalyzing racemization of various amino acids. The enzyme, purified to homogeneity by polyacrylamide gel electrophoresis and ultracentrifugation, has a molecular weight of about 76,000, and is composed of two subunits identical in molecular weight (40,000). The results of enantiomeric analysis of the reaction product and kinetic examination of the reaction indicate that the enzyme catalyzes the complete racemization of substrates. The enzyme has absorption maxima at 280 and 420 nm, and contains 2 mol of pyridoxal 5-phosphate per mol of enzyme. The holoenzyme is resolved to the apoenzyme by treatment with hydroxylamine, and reconstituted by the addition of pyridoxal 5′-phosphate. The very broad substrate specificity of the A. caviae amino acid racemase is comparable to that of the Pseudomonas striata enzyme. However, the...

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.

D-Amino Acid Aminotransferase from a Thermophile, Bacillus SP. YM-1: Enzymological Properties, Cloning of the Gene, and the Amino Acid Sequence

We have isolated a thermophile which grows in a medium containing D-amino acids as a nitrogen source from soil and identified as a new Bacillus species. The bacterium (Bacillus sp. YM-1) showed a very high activity of D-amino acid aminotransferase. The enzyme purified to homogeneity from cell extracts of YM-1 has a molecular weight of about 62,000, and is composed of two subunits identical in molecular weight (30,000). The D-amino acid aminotransferase gene was cloned into Escherichia coli with the vector plasmid pBR322. The clone cells carrying the plasmid of 4.3 kb DNA (pICT113) produced the enzyme as high as 10% of the total cellular proteins. The enzyme overproduced by the clone was purified from cell extracts about 10-fold to homogeneity in about 60% yield by four steps including heat treatment. The complete primary structure of the enzyme involving the position of the active site lysyl residue that binds pyridoxal 5′-phosphate was determined from the nucleotide sequence of the gene and the amino acid sequences of tryptic peptides. The primary structure of D- amino acid aminotransferase shows high sequence homology with that of branched-chain amino acid aminotransferase of E. coli (the ilvE gene product).

A spectrophotometric rate assay of aminoacylase

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.