Bacterial Alanine Racemase Research Articles

Cold-induced aldimine bond cleavage by Tris in Bacillus subtilis alanine racemase.

Pyridoxal 5'-phosphate (PLP) is a versatile cofactor involved in a large variety of enzymatic processes. Most of PLP-catalysed reactions, such as those of alanine racemases (AlaRs), present a common resting state in which the PLP is covalently bound to an active-site lysine to form an internal aldimine. The crystal structure of BsAlaR grown in the presence of Tris lacks this covalent linkage and the PLP cofactor appears deformylated. However, loss of activity in a Tris buffer only occurred after the solution was frozen prior to carrying out the enzymatic assay. This evidence strongly suggests that Tris can access the active site at subzero temperatures and behave as an alternate racemase substrate leading to mechanism-based enzyme inactivation, a hypothesis that is supported by additional X-ray structures and theoretical results from QM/MM calculations. Taken together, our findings highlight a possibly underappreciated role for a common buffer component widely used in biochemical and biophysical experiments.

Bioinformatic analysis of fold-type III PLP-dependent enzymes discovers multimeric racemases.

Pyridoxal-5'-phosphate (PLP)-dependent enzymes are ubiquitous in nature and catalyze a variety of important metabolic reactions. The fold-type III PLP-dependent enzyme family is primarily comprised of decarboxylases and alanine racemases. In the development of a multiple structural alignment database (3DM) for the enzyme family, a large subset of 5666 uncharacterized proteins with high structural, but low sequence similarity to alanine racemase and decarboxylases was found. Compared to these two classes of enzymes, the protein sequences being the object of this study completely lack the C-terminal domain, which has been reported important for the formation of the dimer interface in other fold-type III enzymes. The 5666 sequences cluster around four protein templates, which also share little sequence identity to each other. In this work, these four template proteins were solubly expressed in Escherichia coli, purified, and their substrate profiles were evaluated by HPLC analysis for racemase activity using a broader range of amino acids. They were found active only against alanine or serine, where they exhibited Michaelis constants within the range of typical bacterial alanine racemases, but with significantly lower turnover numbers. As the already described racemases were proposed to be active and appeared to be monomers as judged from their crystal structures, we also investigated this aspect for the four new enzymes. Here, size exclusion chromatography indicated the presence of oligomeric states of the enzymes and a native-PAGE in-gel assay showed that the racemase activity was present only in an oligomeric state but not as monomer. This suggests the likelihood of a different behavior of these enzymes in solution compared to the one observed in crystalline form.

Structural and Functional Adaptation of Vancomycin Resistance VanT Serine Racemases.

ABSTRACTVancomycin resistance in Gram-positive bacteria results from the replacement of the d-alanyl–d-alanine target of peptidoglycan precursors with d-alanyl–d-lactate or d-alanyl–d-serine (d-Ala-d-Ser), to which vancomycin has low binding affinity. VanT is one of the proteins required for the production of d-Ala-d-Ser-terminating precursors by converting l-Ser to d-Ser. VanT is composed of two domains, an N-terminal membrane-bound domain, likely involved in l-Ser uptake, and a C-terminal cytoplasmic catalytic domain which is related to bacterial alanine racemases. To gain insight into the molecular function of VanT, the crystal structure of the catalytic domain of VanTG from VanG-type resistant Enterococcus faecalis BM4518 was determined. The structure showed significant similarity to type III pyridoxal 5′-phosphate (PLP)-dependent alanine racemases, which are essential for peptidoglycan synthesis. Comparative structural analysis between VanTG and alanine racemases as well as site-directed mutagenesis identified three specific active site positions centered around Asn696 which are responsible for the l-amino acid specificity. This analysis also suggested that VanT racemases evolved from regular alanine racemases by acquiring additional selectivity toward serine while preserving that for alanine. The 4-fold-lower relative catalytic efficiency of VanTG against l-Ser versus l-Ala implied that this enzyme relies on its membrane-bound domain for l-Ser transport to increase the overall rate of d-Ser production. These findings illustrate how vancomycin pressure selected for molecular adaptation of a housekeeping enzyme to a bifunctional enzyme to allow for peptidoglycan remodeling, a strategy increasingly observed in antibiotic-resistant bacteria.

Crystal Structure of a Thermostable Alanine Racemase from Thermoanaerobacter tengcongensis MB4 Reveals the Role of Gln360 in Substrate Selection

Pyridoxal 5’-phosphate (PLP) dependent alanine racemase catalyzes racemization of L-Ala to D-Ala, a key component of the peptidoglycan network in bacterial cell wall. It has been extensively studied as an important antimicrobial drug target due to its restriction in eukaryotes. However, many marketed alanine racemase inhibitors also act on eukaryotic PLP-dependent enzymes and cause side effects. A thermostable alanine racemase (AlrTt) from Thermoanaerobacter tengcongensis MB4 contains an evolutionarily non-conserved residue Gln360 in inner layer of the substrate entryway, which is supposed to be a key determinant in substrate specificity. Here we determined the crystal structure of AlrTt in complex with L-Ala at 2.7 Å resolution, and investigated the role of Gln360 by saturation mutagenesis and kinetic analysis. Compared to typical bacterial alanine racemase, presence of Gln360 and conformational changes of active site residues disrupted the hydrogen bonding interactions necessary for proper PLP immobilization, and decreased both the substrate affinity and turnover number of AlrTt. However, it could be complemented by introduction of hydrophobic amino acids at Gln360, through steric blocking and interactions with a hydrophobic patch near active site pocket. These observations explained the low racemase activity of AlrTt, revealed the essential role of Gln360 in substrate selection, and its preference for hydrophobic amino acids especially Tyr in bacterial alanine racemization. Our work will contribute new insights into the alanine racemization mechanism for antimicrobial drug development.

Alanine racemase from Tolypocladium inflatum: A key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity

Cyclosporin A, a cyclic peptide produced by the fungus Tolypocladium inflatum, is a widely employed immunosuppressant drug. Its biosynthesis is strictly dependent on the action of the pyridoxal 5′-phosphate-dependent enzyme alanine racemase, which produces the d-alanine incorporated in the cyclic peptide. This enzyme has a different fold with respect to bacterial alanine racemases. The interest elicited by T. inflatum alanine racemase not only relies on its biotechnological relevance, but also on its evolutionary and structural similarity to the promiscuous enzymes serine hydroxymethyltransferase and threonine aldolase. The three enzymes represent a model of divergent evolution from an ancestral enzyme that was able to catalyse all the reactions of the modern enzymes.A protocol to express and purify with high yield recombinant T. inflatum alanine racemase was developed. The catalytic properties of the enzyme were characterized. Similarly to serine hydroxymethyltransferase and threonine aldolase, T. inflatum alanine racemase was able to catalyse retroaldol cleavage and transamination reactions. This observation corroborates the hypothesis of the common evolutionary origin of these enzymes. A three-dimensional model of T. inflatum alanine racemase was constructed on the basis of threonine aldolase crystal structure. The model helped rationalise the experimental data and explain the catalytic properties of the enzymes.

Characterization of d‐boroAla as a Novel Broad‐Spectrum Antibacterial Agent Targeting d‐Ala‐d‐Ala Ligase

d-boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d-Ala-d-Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d-boroAla was identified and characterized as an antibacterial agent. d-boroAla has activity against both Gram-positive and Gram-negative organisms, with minimal inhibitory concentrations down to 8 μg / mL. A structure-function study on the alkyl side chain (NH(2) -CHR-B(OR')(2) ) revealed that d-boroAla is the most effective agent in a series including boroGly, d-boroHomoAla, and d-boroVal. l-boroAla was much less active, and N-acetylation completely abolished activity. An LC-MS / MS assay was used to demonstrate that d-boroAla exerts its antibacterial activity by inhibition of d-Ala-d-Ala ligase. d-boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d-Ala-d-Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d-Ala-d-Ala ligase. d-boroAla demonstrated a frequency of resistance of 8 × 10(-8) at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d-boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d-Ala-d-Ala ligase targeted antibacterial agents. This study also demonstrates d-boroAla as a possible probe for d-Ala-d-Ala ligase function.

The crystal structure of alanine racemase from Streptococcus pneumoniae, a target for structure-based drug design

BackgroundStreptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens.ResultsHere we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway.ConclusionsWe have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.

Correlation between catalytic activity and monomer–dimer equilibrium of bacterial alanine racemases

From the reaction mechanism and crystal structure analysis, a bacterial alanine racemase is believed to work as a homodimer with a substrate, l-alanine or d-alanine. We analysed oligomerization states of seven alanine racemases, biosynthetic and catabolic, from Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, P. putida and P. fluorescens, with three different methods, gel filtration chromatography, native PAGE and analytical ultracentrifugation. All alanine racemases were proved to be in a dynamic equilibrium between monomeric and dimeric form with every methods used in this study. In both biosynthetic and catabolic alanine racemases, association constants for dimerization were high for the enzymes with high V(max) values. The enzymes with low V(max) values gave the low association constants. We proposed that alanine racemases are classified into two types; the enzymes with low and high-equilibrium association constants for dimerization.

Molecular Characterization of Alanine Racemase in the Kuruma Prawn Marsupenaeus japonicus

Aquatic crustaceans and some bivalve mollusks are known to contain copious amounts of free d-alanine in their tissues. For the first time in the animal kingdom, we have isolated a cDNA clone encoding alanine racemase from the muscle and hepatopancreas of the kuruma prawn Marsupenaeus japonicus. The recombinant enzyme expressed in Escherichia coli exhibited alanine recemase activity. The deduced amino-acid sequence showed only 23-31% identity to bacterial alanine racemases. However, the active site residues and some residues that interact with pyridoxal 5'-phosphate were also conserved in M. japonicus enzyme. There was higher alanine racemase mRNA expression in hepatopancreas than in muscle. In contrast, the d-alanine content in hepatopancreas was lower than that in muscle, suggesting that the physiological functions of free d-alanine may differ among tissues. These data suggest that the alanine racemase gene has been conserved from bacteria to invertebrates throughout a long evolutionary time scale.

D‐Amino acids in the brain: structure and function of pyridoxal phosphate‐dependent amino acid racemases

D-serine serves as a co-agonist of the N-methyl D-aspartate receptor in mammalian brains, and its behavior is probably related to neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis. D-Serine is synthesized by a pyridoxal 5'-phosphate (PLP)-dependent serine racemase. In this minireview, we provide a detailed discussion on the reaction mechanism of the PLP-dependent amino acid racemase on the basis of its 3D structure. We compared the eukaryotic serine racemase with bacterial alanine racemase, the best-studied enzyme among the PLP-dependent amino acid racemases, and thus suggested a putative reaction mechanism for mammalian D-serine synthesis.

A novel zinc-dependent D-serine dehydratase from Saccharomyces cerevisiae

YGL196W of Saccharomyces cerevisiae encodes a putative protein that is unidentified but is predicted to have a motif similar to that of the N-terminal domain of the bacterial alanine racemase. In the present study we found that YGL196W encodes a novel D-serine dehydratase, which belongs to a different protein family from that of the known bacterial enzyme. The yeast D-serine dehydratase purified from recombinant Escherichia coli cells depends on pyridoxal 5'-phosphate and zinc, and catalyses the conversion of D-serine into pyruvate and ammonia with the K(m) and k(cat) values of 0.39 mM and 13.1 s(-1) respectively. D-Threonine and beta-Cl-D-alanine also serve as substrates with catalytic efficiencies which are approx. 3 and 2% of D-serine respectively. L-Serine, L-threonine and beta-Cl-L-alanine are inert as substrates. Atomic absorption analysis revealed that the enzyme contains one zinc atom per enzyme monomer. The enzyme activities toward D-serine and D-threonine were decreased by EDTA treatment and recovered by the addition of Zn2+. Little recovery was observed with Mg2+, Mn2+, Ca2+, Ni2+, Cu2+, K+ or Na+. In contrast, the activity towards beta-Cl-D-alanine was retained after EDTA treatment. These results suggest that zinc is involved in the elimination of the hydroxy group of D-serine and D-threonine. D-Serine dehydratase of S. cerevisiae is probably the first example of a eukaryotic D-serine dehydratase and that of a specifically zinc-dependent pyridoxal enzyme as well.

Cluster analysis of hydration waters around the active sites of bacterial alanine racemase using a 2‐ns MD simulation

Structural data produced by a 2-ns molecular dynamics (MD) simulation on Geobacillus alanine racemase (AlaR; PDB: 1SFT) was used to study hydration around the two AlaR active sites. AlaR is a crucial enzyme for bacterial cell wall biosynthesis. It has been shown previously that the potency of an inhibitor can be increased by incorporating a functional group or atom that displaces hydration sites close to the substrate binding pocket of its target enzyme. The complete linkage algorithm was used for cluster analysis of the active site water positions from 126 solvent configurations sampled at regular intervals from the 2-ns MD simulation. Crystal waters in the 1SFT X-ray structure occupy most of the tightly bound water sites that were discovered. We show here that tightly bound water sites can be identified by cluster analysis of MD-generated coordinates starting with data supplied by a single X-ray structure, and we predict a highly conserved hydration site close to the carboxyl oxygen of L-Ala substrate. This approach holds promise for accelerating the drug design process. We also discuss an analysis of the well-known notion of residence time and introduce a new measure called retention time.

Relationship between Dimerization and Enzyme Activity in Bacterial Alanine Racemases

Relationship between Dimerization and Enzyme Activity in Bacterial Alanine Racemases

Alanine racemase of alfalfa seedlings ( Medicago sativa L.): First evidence for the presence of an amino acid racemase in plants

We demonstrated several kinds of d-amino acids in plant seedlings, and moreover alanine racemase (E.C.5.1.1.1) in alfalfa ( Medicago sativa L.) seedlings. This is the first evidence for the presence of amino acid racemase in plant. The enzyme was effectively induced by the addition of l- or d-alanine, and we highly purified the enzyme to show enzymological properties. The enzyme exclusively catalyzed racemization of l- and d-alanine. The K m and V max values of enzyme for l-alanine were 29.6 × 10 −3 M and 1.02 mol/s/kg, and those for d-alanine are 12.0 × 10 −3 M and 0.44 mol/s/kg, respectively. The K eq value was estimated to be about 1 and indicated that the enzyme catalyzes a typical racemization of both enantiomers of alanine. The enzyme was inactivated by hydroxylamine, phenylhydrazine and some other pyridoxal 5′-phosphate enzyme inhibitors. Accordingly, the enzyme required pyridoxal 5′-phosphate as a coenzyme, and enzymologically resembled bacterial alanine racemases studied so far.

Cloning of alanine racemase genes from Pseudomonas fluorescens strains and oligomerization states of gene products expressed in Escherichia coli

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.

Directed evolution of bacterial alanine racemases with higher expression level

Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the interconversion of L-alanine and D-alanine. It can be classified into two groups: biosynthetic enzymes with low catalytic activity and catabolic enzymes with high catalytic activity. It can react with serine to a limited extent. Two biosynthetic alanine racemase genes in Escherichia coli and Salmonella typhimurium were DNA shuffled, and a very diverse chimeric gene library was constructed. An E. coli serine auxotroph was transformed with the shuffled genes, and the recombinant clones were screened on selective media supplemented with 0.5-5 mM D-serine as an L-serine supplier. Selected clones were expected to contain racemases exhibiting higher catalytic activities toward alanine as well as serine. Three independent clones that grew on selective media were isolated. The specific activities of crude extracts prepared from cells expressing the chimeric racemases were increased up to approximately three times more than those expressing the parental enzymes. The best chimera Ser15 racemase was expressed at a level approximately twofold higher than the parental alanine racemases. This high protein expression was demonstrated to be posttranscriptionally achieved.

Subunit interaction of monomeric alanine racemases from four Shigella species in catalytic reaction

Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer). To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor. The enzymes were eluted on gel filtration as a monomer of about 39 000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine. An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer. The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60 000 and 76 000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively. D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium. The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes. The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands. These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction. Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.

Characterization of an alanine racemase gene from Lactobacillus reuteri.

An alanine racemase gene from Lb. reuteri was cloned by using degenerate oligonucleotides corresponding to conserved regions derived from several bacterial alanine racemases. The protein is 375alphaalpha in length and shows 63.6% homology to the Lb. plantarum alanine racemase. Unlike the single alanine racemase activity found in Lb. plantarum, deletion of the Lb. reuteri alanine racemase reveals a second activity, which is inhibited by beta-chloro- D-alanine.

Functional Genomics with YBL036c.

Functions of more than 2,000 genes in the Saccharomyces cerevisiae genome are still unknown. Gene YBL036c is among them. Gene YBL036c encodes a protein that is PLP (pyridoxalphosphate) dependent. Its sequence does not indicate similarity to any known protein. However, its 3D structure, including the active site residues, are almost identical to that of bacterial alanine racemase. From the structure we can deduce that it is a racemase. An increasing number of data have shown the existence of D enantiomers of amino acids in eukaryotes. D,L-dityrosine was found in spore walls of S. cerevisiae[1] D-serine in mammalian brain cells[2], and an accumulation of some amino-acids in D-form in human tissue, such as bone and eye lens, has been described[3]. Therefore, it seems very interesting and important to recognize a possible racemase activity, which is required for existence and/or amino acid metabolism in eukaryotes.

Functional characterization of alanine racemase from Schizosaccharomyces pombe: a eucaryotic counterpart to bacterial alanine racemase.

Schizosaccharomyces pombe has an open reading frame, which we named alr1(+), encoding a putative protein similar to bacterial alanine racemase. We cloned the alr1(+) gene in Escherichia coli and purified the gene product (Alr1p), with an M(r) of 41,590, to homogeneity. Alr1p contains pyridoxal 5'-phosphate as a coenzyme and catalyzes the racemization of alanine with apparent K(m) and V(max) values as follows: for L-alanine, 5.0 mM and 670 micromol/min/mg, respectively, and for D-alanine, 2.4 mM and 350 micromol/min/mg, respectively. The enzyme is almost specific to alanine, but L-serine and L-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that of L-alanine, respectively. S. pombe uses D-alanine as a sole nitrogen source, but deletion of the alr1(+) gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway for L-alanine coupled with racemization plays a major role in the catabolism of D-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses L-alanine but not D-alanine as a sole nitrogen source. Moreover, D-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1(+) gene enabled S. cerevisiae to grow efficiently on D-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of D-alanine.