Gene Cloning and Characterization of Alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei

Yokoigawa, Kumio; Hirasawa, Reiko; Ueno, Hiroshi; Okubo, Yoko; Umesako, Seiichi; Soda, Kenji

doi:10.1006/bbrc.2001.5817

Cited by 17 publications

(13 citation statements)

References 43 publications

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“…The K m values of the isoleucine 2-epimerase were 5.00 mM for L-Ile and 13.2 mM for D-allo-Ile. These values are relatively high compared with their estimated concentrations in the bacterial cells, but the similar K m values have been reported in bacterial AlaRs (39)(40)(41)(42) and glutamate racemase (29). Until now, the details about the production mechanisms of D-BCAAs in Lactobacillus species have been unclear.…”

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confidence: 62%

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Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

et al. 2013

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Accumulation of D-leucine, D-allo-isoleucine, and D-valine was observed in the growth medium of a lactic acid bacterium, Lactobacillus otakiensis JCM 15040, and the racemase responsible was purified from the cells and identified. The N-terminal amino acid sequence of the purified enzyme was GKLDKASKLI, which is consistent with that of a putative ␥-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative ␥-aminobutyrate aminotransferase gene from L. buchneri JCM 1115 was expressed in recombinant Escherichia coli and then purified to homogeneity. The enzyme catalyzed the racemization of a broad spectrum of nonpolar amino acids. In particular, it catalyzed at high rates the epimerization of L-isoleucine to D-allo-isoleucine and D-allo-isoleucine to L-isoleucine. In contrast, the enzyme showed no ␥-aminobutyrate aminotransferase activity. The relative molecular masses of the subunit and native enzyme were estimated to be about 49 kDa and 200 kDa, respectively, indicating that the enzyme was composed of four subunits of equal molecular masses. The K m and V max values of the enzyme for L-isoleucine were 5.00 mM and 153 mol·min ؊1 ·mg ؊1 , respectively, and those for D-allo-isoleucine were 13.2 mM and 286 mol·min ؊1 ·mg ؊1 , respectively. Hydroxylamine and other inhibitors of pyridoxal 5=-phosphate-dependent enzymes completely blocked the enzyme activity, indicating the enzyme requires pyridoxal 5=-phosphate as a coenzyme. This is the first evidence of an amino acid racemase that specifically catalyzes racemization of nonpolar amino acids at the C-2 position.

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confidence: 62%

“…Enzyme assays were carried out under the standard assay conditions using the recombinant enzyme (1.3 g/ml) and L-Ile (1.25, 2.5, 5, 10, 20, 40, and 60 mM) or D-allo-Ile (5,10,20,40,60,80, and 100 mM). The reaction was stopped after 5 min, when less than 10% of the substrate was consumed.…”

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confidence: 99%

Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

et al. 2013

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“…Bacterial alanine racemases are classified as monomeric (13,15,18) or homodimeric (3,11,12) based on the structure of their subunits. SDS-PAGE revealed that the molecular mass of the purified rAlr of B. anthracis was≈45 kDa.…”

Section: Determination Of the Molecular Mass Of Ralrmentioning

confidence: 99%

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Kanodia¹,

Agarwal²,

Singh³

et al. 2009

BMB Reports

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Alanine racemase catalyzes the interconversion of L-alanine and D-alanine and plays a crucial role in spore germination and cell wall biosynthesis. In this study, alanine racemase produced by Bacillus anthracis was expressed and purified as a monomer in Escherichia coli and the importance of lysine 41 in the cofactor binding octapeptide and tyrosine 270 in catalysis was evaluated. The native enzyme exhibited an apparent Km of 3 mM for L-alanine, and a Vmax of 295 μmoles/min/mg, with the optimum activity occurring at 37 o C and a pH of 8-9. The activity observed in the absence of exogenous pyridoxal 5 -phosphate suggested that the cofactor is bound to the enzyme. Additionally, the UV-visible absorption spectra indicated that the activity was pH independece, of VV-visible absorption spectra suggests that the bound PLP exists as a protonated Schiff's base. Furthermore, the loss of activity observed in the apoenzyme suggested that bound PLP is required for catalysis. Finally, the enzyme followed non-competitive and mixed inhibition kinetics for hydroxylamine and propionate with a Ki of 160 μM and 30 mM, respectively. [BMB reports 2009; 42(1): 47-52]

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“…While the alanine racemases from Salmonella enterica serovar Typhimurium (1,15), Thermus thermophilus (8), and Shigella sp. (19) have been described as monomers, the crystal structure of the alanine racemase from Bacillus stearothermophilus reveals a dimer with two identical polypeptide chains (9). The dimer has two active sites, each of which contains residues from both monomers.…”

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Mutant Analysis Shows that Alanine Racemases from Pseudomonas aeruginosa and Escherichia coli Are Dimeric

Strych

Benedik

2002

J Bacteriol

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Alanine racemases are ubiquitous prokaryotic enzymes providing the essential peptidoglycan precursor D-alanine. We present evidence that the enzymes from Pseudomonas aeruginosa and Escherichia coli function exclusively as homodimers. Moreover, we demonstrate that expression of a K35A Y235A double mutation of dadX in E. coli suppresses bacterial growth in a dominant negative fashion.In most bacteria the D-alanine-D-alanyl dipeptide is essential for cross-linking within the peptidoglycan cell wall. It is produced from the natural amino acid L-alanine by the subsequent action of two enzymes, alanine racemases, to create D-alanine and D-alanine-D-alanyl ligase. While some organisms contain only one alanine racemase, others have two isozymes, encoded by the alr and the dadX genes. The latter group includes Escherichia coli (4) and Pseudomonas aeruginosa (12). Little is known about the advantages or disadvantages of having two enzymes or about the interaction between the enzymes. alr expression is constitutive and seems to provide the D-alanine necessary to maintain cell growth, while dadX gene expression is induced by L-alanine to a level much greater than that of alr and thus is most active when L-alanine is used as a carbon and energy source (4).Alanine racemase is an attractive target for the development of new antibiotics; the enzyme is ubiquitous among bacteria and generally absent in higher eukaryotes, although it has recently been found to function as a means of osmoregulation in some lower marine eukaryotes (7, 10).There are still open questions regarding the phylogenetic history of the two alanine racemase genes. For instance, the sequence identity between Alr and DadX enzymes from the same organism is not significantly greater than between enzymes from different bacteria (12). Likewise, the structural differences between alanine racemases deserve further attention. While the alanine racemases from Salmonella enterica serovar Typhimurium (1, 15), Thermus thermophilus (8), and Shigella sp. (19) have been described as monomers, the crystal structure of the alanine racemase from Bacillus stearothermophilus reveals a dimer with two identical polypeptide chains (9). The dimer has two active sites, each of which contains residues from both monomers. It is noteworthy that, especially within these active center domains, alanine racemase protein sequences from different bacteria share a high level of conservation. Pyridoxal-5Ј-phosphate (PLP), the essential cofactor of alanine racemases, is in aldimine linkage to an N-terminal lysine residue as a protonated Schiff's base (6). This residue (K39), together with a tyrosine residue near the C terminus (Y265) from the other subunit in the dimer, has been identified as being critical for the racemization of alanine (16,17). The contribution of these two residues to enzyme activity was further confirmed through cocrystallization of the B. stearothermophilus alanine racemase with one of its inhibitors, 1-aminoethylphosphonate (11). While the lysine residue is clearly the cat...

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Gene Cloning and Characterization of Alanine Racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei

Cited by 17 publications

References 43 publications

Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

Identification, Purification, and Characterization of a Novel Amino Acid Racemase, Isoleucine 2-Epimerase, from Lactobacillus Species

Biochemical characterization of Alanine racemase- a spore protein produced by Bacillus anthracis

Mutant Analysis Shows that Alanine Racemases from Pseudomonas aeruginosa and Escherichia coli Are Dimeric

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