Hidehiko Tanaka

A versatile bacterial enzyme: l-methionine γ-lyase

Abstract The properties and application of l -methionine γ-lyase [methioninase, l -methionine methanethiol-lyase (deaminating), EC 4.4.1.11], a pyridoxal 5′-phosphate enzyme, purified from Pseudomonas putida and Aeromonas sp. are presented. The enzyme has multicatalytic functions: it catalyses α,γ-elimination and γ-replacement reactions of l -methionine and its analogues (e.g. ethionine, homocysteine, O -acetylhomoserine and selenomethionine), α,β-elimination and β-replacement reactions of l -cysteine and its analogues (e.g. S -methylcysteine, O -acetylserine and Se -methylselenocysteine), deamination and γ-addition of vinylglycine, and deuterium labelling at the α and β positions of l -methionine and other straight-chain l -amino acids. These reactions are applicable to the synthesis of various optically active sulphur and selenium amino acids, preparation of deuterium or tritium labelled l -amino acids, and determination of sulphur amino acids. In addition, the enzyme shows potent anti-neoplastic activity .

Purification of bacterial L-methionine γ-lyase

Abstract A rapid procedure for the purification of l -methionine γ-lyase from Pseudomonas putida ICR 3460 by DEAE-TOYOPEARL 650M and DEAE-Sephadex A-50 column chromatography is presented. The enzyme was purified with an average yield of 75% and showed about 10-fold higher specific activity than the enzyme from P. putida (= P. ovalis ) IFO 3738 reported previously ( H. Tanaka, N. Esaki, and K. Soda (1976) FEBS Lett. 66 , 307–311). The present enzyme has a molecular weight of about 172,000 and consists of four subunits with identical molecular weights (43,000). It shows the typical absorption spectrum of pyridoxal enzyme with maxima at 278 and 420 nm, and contains 4 mol of pyridoxal 5′-phosphate per mole of enzyme. The enzyme has a multicatalytic function similar to the enzyme of P. putida IFO 3738 ( K. Soda, H. Tanaka, and N. Esaki (1983) Trends Biochem. Sci. 8 , 214–217).

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%.

A simple and efficient method for the oligonucleotide-directed mutagenesis using plasmid DNA template and phosphorothioate-modified nucleotide.

We have developed a simple and efficient method for oligonucleotide-directed mutagenesis with double-stranded (plasmid) DNA as a template. The template was simply and rapidly prepared by cell lysis and the following DNA denaturation with alkali. The chain elongation was performed with phosphorothioate-modified nucleotide at 37 degrees C. After the selective digestion of original DNA with NciI and exonuclease III, the desired mutated gene was obtained at a high frequency (about 70%).

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.

Multifunctional biocatalysis: methionine γ-lyase

Abstract Methionine γ-lyase, a pyridoxal 5′-phosphate-dependent enzyme, which is regarded as a key enzyme in bacterial methionine metabolism has multicatalytic function. This article describes the function of the enzyme, with emphasis on its application to synthesis of sulfur and selenium amino acids.

Gene cloning, purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus

Abstract The alanine dehydrogenase ( l -alanine: NAD + oxidoreductase, deaminating, EC 1.4.1.1) gene of Bacillus stearothermophilus IFO12550 was cloned and expressed in Escherichia coli C600 with a recombinant plasmid, pICD301, which was constructed from pBR322 and the alanine dehydrogenase gene derived from B. stearothermophilus . The enzyme overproduced in the clone was purified about 30 fold to homogeneity by heat treatment and two subsequent steps with a yield of 46%. The enzyme of E. coli -pICD301 was immunochemically identical with that of B. stearothermophilus . The enzyme has a molecular weight of about 240,000 and consists of six subunits identical in molecular weight (40,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 30 min; at 55°C and various pHs between 6.0 and 11.5 for 10 min. The enzymological properties are very similar to those of the mesophilic B. sphaericus enzyme (Ohshima, T. and Soda, K., Eur. J. Biochem., 100, 29–39, 1979) except for thermostability.

Enzymatic synthesis of Se-substituted L-selenocysteine with tryptophan synthase

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Mechanism of reactions catalyzed by selenocysteine β-lyase

Abstract The reaction mechanism of selenocystine β-lyase has been studied and it was found that elemental selenium is released enzymatically from selenocysteine, and reduced to H2Se nonenzymatically with dithiothreitol or some other reductants that are added to prepare selenocysteine from selenocystine in the anaerobic reaction system. 1H and 13C NMR spectra of l -alanine formed in 2H2O have shown that an equimolar amount of [β-2H1]- and [β-2H2]alanines are produced. The deuterium isotope effect at the α position was observed; k H k D = 2.4 . These results indicated that the α hydrogen of selenocysteine was removed by a base at the active site, and was incorporated into the α position of alanine, a product, without exchange of a solvent deuterium. When the enzyme was incubated with l -selenocysteine in the absence of added pyridoxal 5′-phosphate, the activity decreased with prolonged incubation time. However, the activity was recovered by addition of 5′-phosphate. The spectrophotometric study showed that the inactivated enzyme was the apo form. The apoenzyme was activated by a combination of pyridoxamine 5′-phosphate and various α-keto acids such as α-ketoglutarate and pyruvate. Thus, the enzyme is inactivated through transamination between selenocysteine and the bound pyridoxal 5′-phosphate to produce pyridoxamine 5′-phosphate and a keto acid derived from selenocysteine. The pyridoxal enzyme, an active form, is regenerated by addition of α-keto acids. This regulatory mechanism is analogous to those of aspartate β-decarboxylase [EC 4.1.1.12], arginine racemase [EC 5.1.1.9], and kynureninase [EC 3.7.1.3] [ K. Soda and K. Tanizawa (1979) Adv. EnZymol.49, 1 ].

Synthesis of L-selenodjenkolate and its degradation with methionine γ-lyase

Abstract A convenient method for the synthesis of a new selenium-containing amino acid, l -selenodjenkolate (3,3′-methylenediselenobis(2-aminopropionic acid)), is described. The starting material, selenocystine, was found to be prepared easily from commercially available β-chloro- l -alanine, elemental selenium, and sodium borohydride. These synthetic procedures are useful for the preparation of the isotope-labeled compounds. The physicochemical properties of selenodjenkolate thus prepared are reported. This amino acid undergoes α,β-elimination to produce pyruvate, formaldehyde, ammonia, and selenium by bacterial methionine γ-lyase under acrobic conditions: the V max and K m values were determined to be 0.5 μmol/min/mg and 2.3 m m , respectively.