Nucleotide sequence of the Bacillus licheniformis ATCC 10716 dat gene and comparison of the predicted amino acid sequence with those of other bacterial species.
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The gene encoding the D-aminotransferase from Bacillus licheniformis was cloned and the complete DNA sequence was determined. The deduced D-aminotransferase protein sequence, consists of 283 amino acids and shows a high degree of homology with other Bacillus D-aminotransferases, branched chain aminotransferase of Escherichia coli and the 4-amino-benzoate-4-deoxychorismate lyase of Bacillus subtilis and Escherichia coli. (+info)
Three-dimensional structure of Escherichia coli branched-chain amino acid aminotransferase at 2.5 A resolution.
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The X-ray crystallographic structure of the branched-chain amino acid aminotransferase from Escherichia coli was determined by means of isomorphous replacement using the selenomethionyl enzyme as one of the heavy atom derivatives. The enzyme is a homo hexamer with D3 symmetry, and the polypeptide chain of the subunit is folded into two domains (small and large domains). The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. The active site structure shows that the following sites can recognize branched-chain amino acids and glutamate as substrates: (1) a hydrophobic core formed by Phe36, Tyr164, Tyr31*, and Val109* for a branched-chain; (2) Arg97 for an acidic side chain of glutamate; and (3) Tyr95 and two main chain NH groups of Thr257 and Ala258 for the alpha-carboxylate of substrates. Although the main chain conformation of the active site is homologous to that of D-amino acid aminotransferase, many of the active site residues are different between them. (+info)
Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.
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We describe a simple method for enzymatic synthesis of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. L-amino acids were produced with thermostable L-amino acid dehydrogenase and formate dehydrogenase (FDH) from alpha-keto acids and ammonium formate with only an intracellular pool of NAD+ for the regeneration of NADH. We constructed plasmids containing, in addition to the FDH gene, the genes for amino acid dehydrogenases, including i.e., leucine dehydrogenase, alanine dehydrogenase, and phenylalanine dehydrogenase. L-Leucine, L-valine, L-norvaline, L-methionine, L-phenylalanine, and L-tyrosine were synthesized with the recombinant E. coli cells with high chemical yields (> 80%) and high optical yields (up to 100% enantiomeric excess). Stereospecific conversion of various alpha-keto acids to D amino acids was also examined with recombinant E. coli cells containing a plasmid coding for the four heterologous genes of the thermostable enzymes D-amino acid aminotransferase, alanine racemase, L-alanine dehydrogenase, and FDH. Optically pure D enantiomers of glutamate and leucine were obtained. (+info)
Compensation for D-glutamate auxotrophy of Escherichia coli WM335 by D-amino acid aminotransferase gene and regulation of murI expression.
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D-glutamate, an indispensable component of peptidoglycans of bacteria, is provided by glutamate racemase in E. coli cells. Compensation for D-glutamate auxotrophy of E. coli WM335 cells lacking the glutamate racemase gene, murI, with the D-amino acid aminotransferase gene suggests that presence of a threshold concentration for the D-glutamate required by E. coli cells, as well as a regulation system for murI expression. (+info)
Characterization of the genes encoding D-amino acid transaminase and glutamate racemase, two D-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208.
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In Bacillus sphaericus and other Bacillus spp., D-amino acid transaminase has been considered solely responsible for biosynthesis of D-glutamate, an essential component of cell wall peptidoglycan, in contrast to the glutamate racemase employed by many other bacteria. We report here the cloning of the dat gene encoding D-amino acid transaminase and the glr gene encoding a glutamate racemase from B. sphaericus ATCC 10208. The glr gene encodes a 28. 8-kDa protein with 40 to 50% sequence identity to the glutamate racemases of Lactobacillus, Pediococcus, and Staphylococcus species. The dat gene encodes a 31.4-kDa peptide with 67% primary sequence homology to the D-amino acid transaminase of the thermophilic Bacillus sp. strain YM1. (+info)
Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 A resolution: implication of the structural role of Leu201 in transamination.
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The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'-phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme. (+info)
Construction and properties of a fragmentary D-amino acid aminotransferase.
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D-Amino acid aminotransferase [EC 2.6.1.21] catalyzes the inter-conversion between various D-amino acids and alpha-keto acids. The subunit of the homodimeric enzyme from Bacillus sp. YM-1 consists of two domains connected by a single loop, which has no direct contact with the active site residues or the cofactor, pyridoxal 5'-phosphate [Sugio, S., Petsko, G.A., Manning, J.M., Soda, K., and Ringe, D. (1995) Biochemistry 34, 9661-9669]. We constructed two plasmids, one encoding a polypeptide fragment corresponding to the N-terminal domain, and the other a fragment corresponding to the C-terminal domain. When both polypeptide fragments were expressed together in the same host cell, an active fragmentary enzyme consisting of two sets of the two polypeptide fragments was produced. When the two polypeptide fragments were expressed separately, each of them provided a soluble protein but with no activity. However, D-amino acid aminotransferase activity appeared upon incubation of a mixture of the two fragments. The active fragmentary enzyme was purified to homogeneity and characterized; it was found to be similar to the wild-type enzyme in various enzymological properties except substrate specificity, inhibition by alpha-ketoglutarate, and thermostability. The fragmentary enzyme showed higher catalytic activity toward several substrates, such as D-lysine and D-arginine, than the wild-type enzyme. (+info)