Transamination As a Side-Reaction Catalyzed by Alanine Racemase of Bacillus Stearothermophilus 1
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J. Biochem. 124, 1163-1169 (1998) Transamination as a Side-Reaction Catalyzed by Alanine Racemase of Bacillus stearothermophilus 1 Yoichi Kurokawa,* Akira Watanabe,* Tohru Yoshimura ,* Nobuyoshi Esaki,*,2 and K enji Soda•õ *Institute for Chemical Research , Kyoto University, Gokasho, Uji, Kyoto 611-0011; and •õFaculty of Engineering, Kansai University, Yamate-cho, Suita , Osaka 564-8680 Received for publication, July 21, 1998 The pyridoxal form of alanine racemase of Bacillus stearothermophilus was converted to the pyridoxamine form by incubation with its natural substrate , D- or L-alanine, under acidic conditions: the enzyme loses its racemase activity concomitantly. The pyridoxamine form of the enzyme returned to the pyridoxal form by incubation with pyruvate at alkaline pH. Thus, alanine racemase catalyzes transamination as a side function. In fact, the apo-form of the enzyme abstracted tritium from [4•Œ-3H]pyridoxamine in the presence of pyruvate. A mutant enzyme containing alanine substituted for Lys39, whose s-amino group forms a Schiff base with the C4•Œ aldehyde of pyridoxal 5•Œ-phosphate in the wild-type enzyme, was inactive as a catalyst for racemization as well as transamination. However, when methylamine was added to the mutant enzyme, it became active in both reactions . These results suggest that the e-amino group of Lys39 participates in both racemization and transamination when catalyzed by the wild-type enzyme. Key words: active site lysine, alanine racemase, pyridoxal 5•Œ-phosphate, reaction mecha nism, transamination. Alanine racemase [EC 5.1.1.1] requires pyridoxal 5•Œ-phos tion occurs produces an antipodal aldimine (D). An isomer phate (PLP) as a coenzyme and catalyzes the interconver ized amino acid and a PLP form of the enzyme are generat sion between L- and D-alanine. The enzyme provides D-ala ed by the subsequent hydrolysis of the aldimine complex nine, which is an indispensable component of the peptido (A). We have proposed that two different bases participate glycan layer of bacterial cell walls (1). We have cloned the in catalysis: one abstracts the a -hydrogen from the sub gene for thermostable alanine racemase from Bacillus strate, and the other returns hydrogen to the deprotonated stearothermophilus, purified the enzyme, and determined intermediate (6). X-ray crystallographic studies have its primary structure (2, 3). The three-dimensional struc suggested that Tyr265 and Lys39, the PLP-binding lysyl ture of the enzyme was also clarified recently (4). residue, serve as catalytic bases in alanine racemase from Reactions catalyzed by PLP-dependent amino acid race B. stearothermophilus (4, 7). mases probably proceed through the mechanism described In addition to aminotransferases, various PLP-enzymes below (4, 5). PLP bound with the active-site lysyl residue such as amino acid decarboxylases and lyases catalyze the through an internal Schiff base (Fig. 1A) reacts with a transamination as a sidereaction (8). We have found that substrate to form an external Schiff base (B). The subse alanine racemase also catalyzes transamination in the same quent a-hydrogen abstraction results in the formation of a manner as amino acid racemase with low substrate speci resonance-stabilized anionic intermediate (C), Reprotona ficity and arginine racemase (9). Transamination is charac tion at C-2 of the substrate moiety on the opposite face of terized by hydrogen transfer between the C-2 of the the planar intermediate to that where the proton abstrac substrate and the C-4•Œ of the cofactor. The transamination catalyzed by amino acid racemases is probably attained 1 This work was supported in part by a Grant-in-Aid for Scientific through a sequence A•¨B•¨C•¨E (or F)•¨G (Fig. 1). An Research, 08680683 (to T.Y.), from the Ministry of Education, equivalent route can be delineated for the antipode: A•¨D Science, Sports and Culture of Japan, and a Research Grant from the •¨C•¨E (or F)•¨G. Japan Society for the Promotion of Science (Research for the Future). In transamination catalyzed by aminotransferases such 2 To whom correspondence should be addressed. E-mail: esaki @ scl. as aspartate aminotransferase (10, 11) and D-amino acid kyoto-u.ac.jp Abbreviations: Bis-tris, 2,2-bis[tris(hydroxymethyl).2,2•Œ,2"-nitrilo aminotransferase (12, 13), the hydrogen transfer between ethanol]; Bis-tris-propane,1,3-bis[tris(hydroxymethyl)methylarnino]- a substrate and PLP is considered to be mediated by the propane; CAPS, 3-(cyclohexylamino)propionesulfonic acid; CHES, lysine residue forming the Schiff base with PLP. We have 2-(cyclohexylamino)ethanesulfonic acid; DAAT, n-amino acid amino examined the role of Lys39 of alanine racemase in trans transferase; PLP, pyridoxal 5•Œ-phosphate; PMP, pyridoxamine 5•Œ- amination by means of site-directed mutagenesis and phosphate; SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)- chemical rescue experiments. We here show that Lys39 aminomethane. plays an essential role in transamination. (C) 1998 by The Japanese Biochemical Society. Vol. 124, No. 6, 1998 1.163 1164 Y. Kurokawa et al. Fig. 1. Probable reaction mechanism of alanine racemase. An through D or B. However, if the C4•Œ-position of the cofactor moiety of internal Schiff base (A) is formed between the ƒÃ-amino group of Lys39 C is protonated, then transamination (e.g., formation of G and ƒ¿-keto and the C4•Œ aldehyde group of PLP. The ƒ¿-hydrogen of a substrate acid) is accomplished through E or F. Tritium is liberated without amino acid is removed from intermediate B or D to form a quinoid discrimination from both [4•ŒR-3H] and [4•ŒS-3H]PMP in the transami intermediate, C. If a proton is returned to the a-position of the nation catalyzed by the apo-enzyme with an ƒ¿-keto acid. Therefore, G substrate moiety of intermediate C, then racemization is accomplished is converted to A through E or F as an equivalent intermediate. with the AccI-AccI linker into the EcoRI-AccI sites of MATERIALS AND METHODS vector pUC119. ssDNA was prepared from the E. coli BW313 transformed with pAR420 and infected with Materials-The plasmid pMDalr3 carrying the alanine M13KO7 phage. It was used as a template for the site- racemase gene from B. stearothermophilus was prepared as directed mutagenesis. Two oligonucleotides, 5•Œ-ATTATG- described previously (14). Phagemid vectors pUC118, GCGGTCGTGGCAGCGAACGCCTATGGA- 3•Œ and 5•Œ-CT- pUC119, helper phage M13KO7, E. coli JM109, BW313, GGTGAGTATTCAACCAAGTC-3•Œ, were synthesized by BMH71-18 mutS, restriction nucleases, T4 DNA polymer the phosphoamidite method and used for the replacement ase, T4 DNA ligase, T4 DNA kinase, and calf alkaline of Lys39 by an alanyl residue and the elimination of Scal phosphatase were purchased from Takara Shuzo, Kyoto. A site of the pUC119 vector, respectively. Site-directed mixture of dNTPs was purchased from Boehringer Mann mutagenesis was carried out by Kunkel's method (16) in heim, Germany. Alanine dehydrogenase was a gift from Dr. combination with the USE (Unique-Site Elimination) H. Kondo of Unitika, Osaka. D-Amino acid aminotransfer method (17). Mutation was confirmed by DNA sequencing ase was prepared as described previously (15). L-Lactate by the dye deoxy terminator method with an Applied dehydrogenase was purchased from Boehringer Mannheim Biosystems Model 373A automated DNA sequencer. The (Germany). Other chemicals were of analytical grade 0.5-kb EcoRI-AccI fragment of the obtained plasmid was commercially available. ligated into the EcoRI-AccI sites of the plasmid pAR310. Site-Directed Mutagenesis-Plasmid pAR310 was con Purification of the Enzymes-The cloned wild-type structed by ligation of the 1.4-kb EcoRI-Hindlll fragment alanine racemase of B. stearothermophilus was purified as from pMDalr3 into the EcoRI-Hindlll sites of vector described previously (2, 14). The mutant enzyme was pUC118. Plasmid pAR420 was constructed by ligation of purified as follows. E. coli JM109 cells were transformed the 0.5-kb EcoRI-AccI fragment from pMDalr3 together with the plasmid pAR310 bearing the mutant alanine J. Biochem. Transamination by Alanine Racemase 1165 racemase gene. Transformant cells were cultivated in 3 solution was determined as the amount of NADH consumed liters of Luria-Bertani's medium containing ampicillin (60 in the assay mixture containing 250 u mol of Bis-Tris ƒÊ g/ml) at 37•Ž for 10 h. The mutant enzyme was produced propane buffer (pH 8.5), 0.16 ƒÊmol of NADH, 5 ƒÊmol of by induction by the addition of 0.1 mM isopropyl-f3-D-thio ƒ¿-ketoglutarate , 2.2 units of D-amino acid aminotransfer galactopyranoside at 2 h after inoculation. Cells were ase, and 5.5 units of lactate dehydrogenase in a final volume harvested by centrifugation at 6,000 rpm for 10 min at 4•Ž of 1.0 ml. After incubating the assay mixture at 37•Ž for 60 and washed with 0.85% NaCl twice. Cells (about 10 g , wet min, the decrease in absorbance at 340 nm was measured. weight) were suspended in 100 ml of 100 mM potassium Preparation of Apoenzymes-The wild-type and mutant phosphate buffer (pH 7.2) containing 20 ƒÊM PLP, 0.01% alanine racemases were converted to apo-forms by dialysis 2-mercaptoethanol, 0.1 mM phenylmethyl-sulfonyl fluo against 100 mM potassium phosphate buffer (pH 7.2) ride, and 0.1 mM p-toluensulfonyl-L-phenylalanine chloro containing 30 mM hydroxylamine and 0.01% 2-mercapto methyl ketone. The purification procedurs described ethanol at 4 for 24 h. The enzyme solutions were then below were carried out at 4•Ž unless otherwise specified. dialyzed against 20 mM potassium phosphate buffer (pH After the cell suspension was sonicated for 20 min, the 7.2) containing 0.01% 2-mercaptoethanol at 4•Ž for 8 h. lysate was centrifuged at 8,000 rpm for 20 min.