SYNTHESIS OF L-[ 15 N]-ISOLEUCINE, L-[ 15 N]-NORLEUCINE AND L-[ 15 N]-NORVALINE BY ENZYMATIC METHOD
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1 SYNTHESIS OF L-[ 15 N]-ISOLEUCINE, L-[ 15 N]-NORLEUCINE AND L-[ 15 N]-NORVALINE BY ENZYMATIC METHOD IULIA LUPAN 1, BEATRIX FERENCZ 1, MARIA CHIRIAC 2, NADIA BUCURENCI 3, OCTAVIAN POPESCU 1 1 Molecular Biology Center, Institute for Interdisciplinary Experimental Research, Babeş-Bolyai University, Str. August Treboniu Laurian 42, Cluj-Napoca, Romania 2 National Institute for Research and Development of Isotopic and Molecular Technologies, Cluj-Napoca, Romania 3 Cantacuzino National Institute of Research and Development for Microbiology and Immunology, Spl. Independenţei 103, Bucharest, Romania (Received September 15, 2006) Leucine dehydrogenase (Leu DH) (EC ) is a NAD-dependent oxidoreductase that catalyses reversible deamination of L-leucine and other aliphatic L-amino acids to their keto analogues. We cloned the structural gene (leudh) of Bacillus stearothermophilus into pet21b vector and expressed in E.coli strain BL21(DE3). The recombinant LeuDH was used for preparing L-[ 15 N]-isoleucine, L-[ 15 N]-norleucine and L-[ 15 N]-norvaline from the corresponding α-keto acids, by a simple procedure. We used a coupled enzymatic system: LeuDH as a catalyst and glucose dehydrogenase for NADH regeneration, using glucose as an electron donor. Reaction efficiencies were between 80 and 95%. Our results demonstrate the utility of recombinant amino acid dehydrogenases for synthesis of 15 N-labeled amino acids and the advantages of the enzymatic method. Key words: 15 N-labeled amino acid, leucine dehydrogenase, enzyme expression, Bacillus stearothermophilus. INTRODUCTION Stable isotope-labeled amino acids, particularly dual- or triple-labeled, can provide extremely high resolution structural information and form an important part of the toolkit for researchers in academia and industry, in a range of applications including: structural determinations of proteins, protein protein and protein drug interaction studies, metabolism and nutritional research, as well as labeled protein synthesis. 15 N-labeled amino acids are commonly commercially unavailable or expensive. The chemical synthesis of amino acids is disadvantageous, because a racemic product is produced. Differently, by the enzymatic method only one isomer (L or D) can be produced. Considerable attention has been paid to multienzyme reaction systems implied in stereospecific production of amino acids (1). ROM. J. BIOCHEM., 43 44, ( )
2 32 Iulia Lupan et al. 2 A method for enzymatic synthesis of L-amino acids from α-keto acids, using NAD(P)-dependent amino acid dehydrogenases, has been proposed (2, 3). Amino acid dehydrogenases are NAD-dependent oxidoreductases which catalyze the reversible deamination of L-amino acids to their keto analogues: L-amino acid + NAD + + H 2 O α-keto acid + NH NADH L-amino acids can be obtained in an almost quantitative manner by the reductive amination of keto acids, because the reaction equilibrium favors amino acid formation. Leucine dehydrogenase (LeuDH) (EC ) catalyzes the reversible deamination of L-leucine and other aliphatic L-amino acids to their keto analogues. The enzyme occurs ubiquitously in Bacillus species and functions catabolically in the bacterial metabolism of branched-chain L-amino acids. The enzyme has been purified to homogeneity from B. sphaericus (4), B. cereus (5), B. caldolyticus (6), and some thermophiles such as B. stearothermophilus (7), B. licheniformis (8), Clostridium thermoaceticum (9) and Thermoactinomyces intermedius (10). Several L-amino acids have been prepared using LeuDH in an enzyme-membrane reactor (11). MATERIAL AND METHODS Molecular biology methods. LeuDH gene from B. stearothermophilus was cloned in pet21b vector. The fragment for leudh gene was amplified by PCR, using the following primers: F1LDHBst 5 -GGTGGATCCGATGGAATTGTTCAAATATATGG-3 RLeBst 5 -TATTCTCGAGTATTGCCGAAGCACCTGC-3. The amplified fragment containing BamHI and XhoI linkers was digested and inserted in pet21b Novagen vector. The recombinant molecules were transformed into E. coli strain DH5α. The screening of the recombinant plasmids isolated from colonies obtained after transformation by electroporation was made by restriction enzyme digest. The cloned gene was sequenced with ABI Prism 310 Genetic Analyzer using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). In order to express the recombinant plasmid, the purified DNA was transferred into strain BL21(DE3). E. coli BL21(DE3) cells carrying recombinant plasmids were cultured at 37ºC in Luria-Bertani medium supplemented with 100 mg/l ampicillin. Biochemical methods. The standard reaction mixture for reductive amination consisted of 50 mm Tris-HCl ph 8.0, 10 mm α-keto acid, 0.15 mm NADH, 1 M NH 4 Cl and bacterial lysate with recombinant leucine dehydrogenase. The standard reaction mixture for glucose oxidation consists of 50 mm Tris-HCl ph 8.0, 10 mm glucose, 2 mm NAD and bacterial lysate with recombinant glucose dehydrogenase.
3 3 [ 15 N]-amino acid synthesis 33 One unit of enzyme was defined as the amount that catalyzed the formation (or consumption) of 1 µmol of NADH per minute under the standard assay conditions. When the coupled enzymatic system was used, the reaction mixture contained: 570 mm glucose, 115 mm keto acid, 115 mm NH 4 Cl (99% 15 N), 7 mm NADH. The excess of glucose was necessary for glucose dehydrogenase stability; it is known that the enzyme is unstable in the absence of glucose. The NH 4 Cl was assayed by the Berthelot method (12). The reaction mixture was stirred at 30 o C adjusting the ph value at 8 with 1N NaOH. Once no further change in ph was observed, the reaction mixture was heated at 85 o C for 15 min. After cooling, the denatured protein was removed by filtration or centrifugation and the reaction solution (50 ml) was loaded into a 120 ml column ( cm) of Dowex 50WX- 8 in H + form. After washing with distilled water, the adsorbed amino acid was eluted with 1 M NH 4 OH. The eluted fractions containing the labeled [ 15 N]-amino acid were concentrated in a rotary vacuum evaporator at 50 o C, to approximately 5 ml. Ninety ml of anhydrous ethanol/ether (50/50, v/v) were added and the mixture was kept overnight in the refrigerator, while the amino acid crystallized. The precipitate was filtered off and dried. RESULTS AND DISCUSSION The enzymatic method is the most simple and used technique to synthesize 15 N- labeled amino acids from keto acids and 15 NH 4 +. The advantage of the enzyme-catalyzed synthesis of amino acids by reductive amination is that it is restricted to the L-enantiomers as products. The corresponding NAD-depending D-amino acid dehydrogenases are not known. The ability of the recombinant leucine dehydrogenase obtained by us to catalyze the reductive amination of various keto acids is shown in Table 1. One can observe that α-ketoisovaleric acid is the preferred substrate. Table 1 Substrate specificities of recombinant leucine dehydrogenase in reductive amination α-keto acid Specific activity (U/mg protein) Relative activity (%) α-ketoisovaleric acid α-keto-β-methyl-n-valeric acid α-ketoisocaproic acid α-ketocaproic acid α-keto-γ-methylthiobutyric acid α-ketobutyric acid
4 34 Iulia Lupan et al. 4
5 5 [ 15 N]-amino acid synthesis 35 We synthesized L-[ 15 N]-isoleucine, L-[ 15 N]-norvaline and L-[ 15 N]-norleucine by reductive amination of DL-α-keto-β-methyl-n-valerate, α-ketovalerate and α-ketocaproate respectively in the presence of 15 NH 4 Cl and NADH and using recombinant LeuDH as a catalyst. As the NADH coenzyme is a very expensive and labile chemical, efficient coenzyme regeneration was needed to make this process economical. The simultaneous regeneration also offers a chance to overcome the stoichiometric relation between product and coenzyme by an efficient internal recycling. So, the reaction mixtures included in all cases glucose dehydrogenase and glucose for NADH regeneration. The ammonia consumption in L-[ 15 N]- isoleucine, L-[ 15 N]-norleucine and L-[ 15 N]-norvaline synthesis is given in Figure 1. Reaction efficiencies were between 80 and 95% (Table 2). Table 2 Reaction efficiencies of labeled amino acid enzymatic syntheses Amino acid Reaction efficiency (%) L-isoleucine 85 L-norleucine 80 L-norvaline 95 We show here the usefulness of recombinant leucine dehydrogenase and glucose dehydrogenase in enzymatic synthesis of 15 N-labelled amino acids. The NADH regeneration is more convenient with glucose dehydrogenase (the enzyme is more active and the substrates do not inhibit the reactions) than other methods described before with formate dehydrogenase (1) or alcohol dehydrogenase (13). Acknowledgements. This research was supported by the BIOTECH (3/260) and CNCSIS (code 1767) grants. REFERENCES 1. A. Galkin, L. Kulakova, T. Yoshimura, K. Soda, N. Esaki, Appl. Environ. Microbiol., 63, (1997). 2. T. Oshima, K. Soda, Int. Ind. Biotechnol., 9, 5 11 (1989). 3. A. Galkin, L. Kulakova, H. Yamamoto, K. Tanizawa, H.T. Tanaka, N. Esaki, K. Soda, J. Ferment. Bioeng., 83, (1997). 4. T. Ohshima, H. Misono, K Soda, J. Biol. Chem., 253, (1978). 5. H. Schutte, W. Hummel, H. Tsai, M.R. Kula, Appl. Microbiol. Biotechnol., 22, (1985). 6. U. Karst, H. Schutte, H. Baydoun, H. Tsai, J. Gen. Microbiol., 135, (1989). 7. T. Ohshima, S. Nagata, K. Soda, Arch. Microbiol., 141, (1985). 8. S. Nagata, S. Bakthavatsalam, A.G. Galkin, H. Asada, S. Sakai, N. Esaki, K. Soda, T. Ohshima, S. Nagasaki, K. Soda, Appl. Microbiol. Biotechnol., 44, (1995).
6 36 Iulia Lupan et al H. Shimoi, S. Nagata, N. Esaki, H. Tanaka, K. Soda, Agric. Biol. Chem., 51, (1987). 10. T. Ohshima, N. Nishida, S. Bakthavatsalam, K. Kataoka, H. Takada, T. Yoshimura, N. Esaki, K. Soda, Eur. J. Biochem., 222, (1994). 11. W. Hummel, M.R. Kula, Eur. J. Biochem., 184, 1-13 (1989). 12. M. Berthelot, Répertoire de Chemie appliqué, 1, 284 (1859). 13. A. Mocanu, G. Niac, A. Ivanof, V. Gorun, N. Palibroda, E. Varga, M. Bologa, O. Bârzu, FEBS Lett., 143, (1982).
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