The first identification and characterization of a histidine-specific amino acid racemase, histidine racemase from a lactic acid bacterium, Leuconostoc mesenteroides subsp. sake NBRC 102480.

We expressed a histidine racemase from Leuconostoc mesenteroides subsp. sake NBRC 102480 (Lm-HisR) successively in a soluble fraction of Escherichia coli BL21 (DE3) and then highly purified it from the cell-free extract. Lm-HisR showed amino acid racemase activity on histidine specifically. This is the first example of an amino acid racemase specifically acting on histidine. Phylogenetic analysis of Lm-HisR showed that Lm-HisR was located far from the cluster of alanine racemases reported thus far and only in lactic acid bacteria of the genus Leuconostoc. Alignment of the primary structure of Lm-HisR with those of lysine and alanine racemases and alanine racemase homologs previously reported revealed that the PLP-binding lysine and catalytic tyrosine were completely conserved, and some residues that are unique to the phylogenetic branch of Lm-HisR, Phe44, Ser45, Thr174, Thr206, His286, Ser287, Phe292, Gly312, Val357, and Ala358 were identified. We determined the crystal structure of Lm-HisR complexed with PLP at a 2.1-Å resolution. The crystal structure contained four molecules (two dimers) in the asymmetric unit. When comparing the 3D structure of Lm-HisR with those of racemases from Geobacillus stearothermophilus and Oenococcus oeni, Met315 was completely conserved, but Val357 was not. In addition, two significant differences were observed between Lm-HisR and G. stearothermophilus alanine racemase. Phe44 and His286 in Lm-HisR corresponded to Tyr43 and Tyr284 in G. stearothermophilus alanine racemase, respectively. Based on the structural analysis, comparison with alanine racemase, and docking simulation, three significant residues, Phe44, His286, and Val357, were identified that may control the substrate specificity of Lm-HisR.

Characterization of d-succinylase from Cupriavidus sp. P4-10-C and its application in d-amino acid synthesis.

d-Amino acids are important building blocks for various compounds, such as pharmaceuticals and agrochemicals. A more cost-effective enzymatic method for d-amino acid production is needed in the industry. We improved a one-pot enzymatic method for d-amino acid production by the dynamic kinetic resolution of N-succinyl amino acids using two enzymes: d-succinylase (DSA) from Cupriavidus sp. P4-10-C, which hydrolyzes N-succinyl-d-amino acids enantioselectively to their corresponding d-amino acid, and N-succinyl amino acid racemase (NSAR, EC.4.2.1.113) from Geobacillus stearothermophilus NCA1503. In this study, DSA and NSAR were purified and their properties were investigated. The optimum temperature of DSA was 50°C and it was stable up to 55°C. The optimum pH of DSA and NSAR was around 7.5. In d-phenylalanine production, the optical purity of product was improved to 91.6% ee from the examination about enzyme concentration. Moreover, 100 mM N-succinyl-dl-tryptophan was converted to d-tryptophan at 81.8% yield with 94.7% ee. This enzymatic method could be useful for the industrial production of various d-amino acids.

Identification and characterization of novel broad-spectrum amino acid racemases from Escherichia coli and Bacillus subtilis.

The peptidoglycan layer of the bacterial cell wall typically contains D-alanine (D-Ala) and D-glutamic acid (D-Glu), and also various non-canonical D-amino acids that have been linked to peptidoglycan remodeling, inhibition of biofilm formation, and triggering of biofilm disassembly. Bacteria produce D-amino acids when adapting to environmental changes as a common survival strategy. In our previous study, we detected non-canonical D-amino acids in Escherichia coli grown in minimal medium. However, the biosynthetic pathways of non-canonical D-amino acids remain poorly understood. In the present study, we identified amino acid racemases in E. coli MG1655 (YgeA) and Bacillus subtilis (RacX) that produce non-canonical D-amino acids other than D-Ala and D-Glu. We characterized their enzymatic properties, and both displayed broad substrate specificity but low catalytic activity. YgeA preferentially catalyzes the racemization of homoserine, while RacX preferentially racemizes arginine, lysine, and ornithine. RacX is dimeric, and appears not to require pyridoxal 5'-phosphate (PLP) as a coenzyme as is the case with YgeA. To our knowledge, this is the first report on PLP-independent amino acid racemases possessing broad substrate specificity in E. coli and B. subtilis.

Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains.

A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe.

Biochemical and Mutational Characterization of N-Succinyl-Amino Acid Racemase from Geobacillus stearothermophilus CECT49.

N-Succinyl-amino acid racemase (NSAAR), long referred to as N-acyl- or N-acetyl-amino acid racemase, is an enolase superfamily member whose biotechnological potential was discovered decades ago, due to its use in the industrial dynamic kinetic resolution methodology first known as "Acylase Process". In previous works, an extended and enhanced substrate spectrum of the NSAAR from Geobacillus kaustophilus CECT4264 toward different N-substituted amino acids was reported. In this work, we describe the cloning, purification, and characterization of the NSAAR from Geobacillus stearothermophilus CECT49 (GstNSAAR). The enzyme has been extensively characterized, showing a higher preference toward N-formyl-amino acids than to N-acetyl-amino acids, thus confirming that the use of the former substrates is more appropriate for a biotechnological application of the enzyme. The enzyme showed an apparent thermal denaturation midpoint of 77.0 ± 0.1 °C and an apparent molecular mass of 184 ± 5 kDa, suggesting a tetrameric species. Optimal parameters for the enzyme activity were pH 8.0 and 55-65 °C, with Co(2+) as the most effective cofactor. Mutagenesis and binding experiments confirmed K166, D191, E216, D241, and K265 as key residues in the activity of GstNSAAR, but not indispensable for substrate binding.

Identification, purification, and characterization of a novel amino acid racemase, isoleucine 2-epimerase, from Lactobacillus species.

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 γ-aminobutyrate aminotransferase from Lactobacillus buchneri. The putative γ-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 γ-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 Km and Vmax values of the enzyme for l-isoleucine were 5.00 mM and 153 µmol·min(-1)·mg(-1), respectively, and those for d-allo-isoleucine were 13.2 mM and 286 µmol·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.

Purification, crystallization and preliminary X-ray crystallographic analysis of diaminopimelate epimerase from Acinetobacter baumannii.

The meso isomer of diaminopimelate (meso-DAP) is a biosynthetic precursor of L-lysine in bacteria and plants, and is a key component of the peptidoglycan layer in the cell walls of Gram-negative and some Gram-positive bacteria. Diaminopimelate epimerase (DapF) is a pyridoxal-5'-phosphate-independent racemase which catalyses the interconversion of (6S,2S)-2,6-diaminopimelic acid (LL-DAP) and meso-DAP. In this study, DapF from Acinetobacter baumannii was overexpressed in Escherichia coli strain SoluBL21, purified and crystallized using a vapour-diffusion method. A native crystal diffracted to a resolution of 1.9 Šand belonged to space group P3(1) or P3(2), with unit-cell parameters a = b = 74.91, c = 113.35 Å, α = ß = 90, γ = 120°. There were two molecules in the asymmetric unit.

Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8.

Glutamic acid racemases (MurI, E.C. 5.1.1.3) catalyse the racemisation of L- and D-glutamic acid. MurIs are essential enzymes for bacterial cell wall synthesis, which requires d-glutamic acid as an indispensable building block. Therefore these enzymes are suitable targets for antimicrobial drugs as well as for the potential design of auxotrophic selection markers. A high expression system in Escherichia coli BL21 (DE3) was constructed to produce and characterise the biochemical properties of the MurI from Lactobacillus plantarum NC8. In a 4-L-bioreactor cultivation, 3266 nkat(D-Glu)/mg(protein) of specific enzyme activity was produced. The recombinant, tag-free Murl was purified by an innovative affinity chromatography method using L-glutamic acid as the relevant docking group, followed by an anion exchange chromatography step (purification factor 9.2, yield 11%). This two-step purification strategy resulted in a Murl sample with a specific activity of 34,060 nkat(D-Glu)/mg(protein), comprising a single protein band in SDS-PAGE. The purified Murl possessed an assay temperature optimum of 50 °C, but it was not stable at this temperature. The half-lives of the purified Murl were 162 h at 20 °C and only 1.9 h at 40 °C. The Murl activity was maximum between pH 7 and 10, resulting in a maximal half-life of 287 h at pH 7. Only D- and L-glutamic acid were recognised as substrates for the Murl with similar K(cat)/K(M) ratios of 3.6s(-1)/mM for each enantiomer.

Substrate specificity of a peptidyl-aminoacyl-L/D-isomerase from frog skin.

In the skin of fire-bellied toads (Bombina species), an aminoacyl-L/D-isomerase activity is present which catalyses the post-translational isomerization of the L- to the D-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position 1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position 3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme. Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides containing D-amino acids.

Determination of 4-hydroxyproline-2-epimerase activity by capillary electrophoresis: A stereoselective platform for inhibitor screening of amino acid isomerases.

Isomerases involved in the metabolism of D/L-amino acids represent promising therapeutic targets for treatment of disease. Herein, we report a tunable platform for the assessment of enzymatic kinetics involving amino acid isomerization by CE that offers improved selectivity and sensitivity over traditional methods. Enzyme activity and competition assays were evaluated for various hydroxyproline diastereoisomers, proline enantiomers and their structural analogs using 4-hydroxyproline-2-epimerase as a model system. In this work, pyrrole 2-carboxylic acid was found to be a selective inhibitor of 4-hydroxyproline-2-epimerase with a half-maximal inhibition concentration of (2.3 + or - 0.1) mM. Reliable methods for unambiguous characterization of amino acid isomerases are required for the screening of novel inhibitors with epimerase and/or racemase activity.

Crystallization and preliminary X-ray diffraction analysis of diaminopimelate epimerase from Escherichia coli.

Diaminopimelate (DAP) epimerase (EC 5.1.1.7) catalyzes the penultimate step of lysine biosynthesis in bacteria and plants, converting L,L-diaminopimelate to meso-diaminopimelate. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DAP epimerase from Escherichia coli are presented. Crystals were obtained in space group P4(1)2(1)2 and diffracted to 2.0 A resolution, with unit-cell parameters a = b = 89.4, c = 179.6 A. Molecular replacement was conducted using Bacillus anthracis DAP epimerase as a search model and showed the presence of two molecules in the asymmetric unit, with an initial R(free) of 0.456 and R(work) of 0.416.

Kinetic characterization and quaternary structure of glutamate racemase from the periodontal anaerobe Fusobacterium nucleatum.

Cofactor-independent glutamate racemases (GRs) that supply the d-glutamate required for biosynthesis of the peptidoglycan that encapsulates bacterial cells are attractive targets for the development of antibacterial drugs. Recombinant GR from Fusobacterium nucleatum (FnGR), a Gram-negative anaerobe involved in periodontal disease, was overproduced, purified, and characterized. Unlike most other GRs, FnGR is a pseudosymmetric enzyme, catalyzing the racemization of glutamate enantiomers with similar kinetic parameters (k(cat)(L-->D)=17.4+/-0.8s(-1), K(m)(L-->D)=1.04+/-0.07mM, k(cat)(D-->L)=26+/-1s(-1), and K(m)(D-->L)=1.7+/-0.1mM; [corrected] pH optimum approximately 8.5). Mutational analysis of residue 151 (A151V) located at the entryway to the active site revealed that FnGR is very sensitive to increased steric bulk at this position. Blue native-polyacrylamide gel electrophoresis, Ferguson plot analyses, and cross-linking studies, indicated that FnGR existed predominately as dimers. Unlike Bacillus subtilis GR, the presence of glutamate did not significantly alter the position of the monomer-dimer equilibrium of FnGR.

Enzyme-assisted physicochemical enantioseparation processes-Part III: Overcoming yield limitations by dynamic kinetic resolution of asparagine via preferential crystallization and enzymatic racemization.

The application of enantioseparation methods alone can only yield up to 50% of the desired chiral product. Thus enantioseparation becomes more attractive when accompanied by the racemization of the counter-enantiomer. Here we present first results of dynamic kinetic resolution of L-asparagine (L-Asn) via preferential crystallization and enzymatic racemization from a racemic, supersaturated solution on a 20 mL scale. An enzyme lyophilisate (WT amino acid racemase from P. putida KT2440 (E.C. 5.1.1.10), overexpressed in E. coli BL21(DE3)) was used for in situ racemization (enzyme concentrations varying from 0 to 1 mg/mL). When preferential crystallization was applied without any enzyme, a total of 31 mg of L-Asn monohydrate could be crystallized, before crystal formation of d-Asn started. Crystallization experiments accompanied by enzymatic racemization led to a significant increase of crystallized L-Asn (198 mg L-Asn monohydrate; >92%ee) giving the first experimental proof for this new process concept of dynamic kinetic resolution via preferential crystallization and enzymatic racemization. Measurements of the racemase activity before and after the crystallization process showed no significant differences, which would allow for enzyme recovery and recycling.

A periplasmic, pyridoxal-5'-phosphate-dependent amino acid racemase in Pseudomonas taetrolens.

The pyridoxal-5'-phosphate (PLP)-dependent amino acid racemases occur in almost every bacterium but may differ considerably with respect to substrate specificity. We here isolated the cloned broad substrate specificity racemase ArgR of Pseudomonas taetrolens from Escherichia coli by classical procedures. The racemase was biochemically characterized and amongst other aspects it was confirmed that it is mostly active with lysine, arginine and ornithine, but merely weakly active with alanine, whereas the alanine racemase of the same organism studied in comparison acts on alanine only. Unexpectedly, sequencing the amino-terminal end of ArgR revealed processing of the protein, with a signal peptide cleaved off. Subsequent localization studies demonstrated that in both P. taetrolens and E. coli ArgR activity was almost exclusively present in the periplasm, a feature so far unknown for any amino acid racemase. An ArgR-derivative carrying a carboxy-terminal His-tag was made and this was demonstrated to localize even in an E. coli mutant devoid of the twin-arginine translocation (Tat) pathway in the periplasm. These data indicate that ArgR is synthesized as a prepeptide and translocated in a Tat-independent manner. We therefore propose that ArgR translocation depends on the Sec system and a post-translocational insertion of PLP occurs. As further experiments showed, ArgR is necessary for the catabolism of D: -arginine and D: -lysine by P. taetrolens.

Determinants of catalytic power and ligand binding in glutamate racemase.

Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D- and L-glutamate and are important for viability in several gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the C alpha of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from C alpha, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.

Inhibition of DNA gyrase activity by Mycobacterium smegmatis MurI.

Glutamate racemase (MurI) catalyzes the interconversion of l-glutamate to d-glutamate, one of the essential amino acids present in the peptidoglycan. In addition to this essential enzymatic function, MurI from Escherichia coli, Bacillus subtilis and Mycobacterium tuberculosis inhibit DNA gyrase activity. A single gene for murI found in the Mycobacterium smegmatis genome was cloned and overexpressed in a homologous expression system to obtain a highly soluble enzyme. In addition to the racemization activity, M. smegmatis MurI inhibits DNA gyrase activity by preventing DNA binding of gyrase. The sequestration of the gyrase by MurI results in inhibition of all reactions catalyzed by DNA gyrase. More importantly, MurI overexpression in vivo in mycobacterial cells provides protection against the action of ciprofloxacin. The DNA gyrase-inhibitory property thus appears to be a typical characteristic of MurI and would have probably evolved to either modulate the function of the essential housekeeping enzyme or to provide protection to gyrase against gyrase inhibitors, which cause double-strand breaks in the genome.

Characterization and isolation of L-to-D-amino-acid-residue isomerase from platypus venom.

Platypus venom contains an isomerase that reversibly interconverts the second amino-acid residue in some peptides between the L-form and the D-form. The enzyme acts on the natriuretic peptides OvCNPa and OvCNPb, and on the defensin-like peptides DLP-2 and DLP-4, but it does not act on DLP-1. While the isomerization of DLP-2 to DLP-4 is inhibited by the amino-peptidase inhibitor amastatin, it is not affected by the leucine amino-peptidase inhibitor bestatin. The enzyme, that is only present in minute quantities in an extract of the venom gland, is thermally stable up to 55 degrees C, and it was found by anion-exchange chromatography to be acidic. Isolation of the isomerase was carried out by combined ion-exchange chromatography and reverse-phase high performance liquid chromatography (HPLC).

Glutamate racemase from Mycobacterium tuberculosis inhibits DNA gyrase by affecting its DNA-binding.

Glutamate racemase (MurI) catalyses the conversion of l-glutamate to d-glutamate, an important component of the bacterial cell wall. MurI from Escherichia coli inhibits DNA gyrase in presence of the peptidoglycan precursor. Amongst the two-glutamate racemases found in Bacillus subtilis, only one inhibits gyrase, in absence of the precursor. Mycobacterium tuberculosis has a single gene encoding glutamate racemase. Action of M.tuberculosis MurI on DNA gyrase activity has been examined and its mode of action elucidated. We demonstrate that mycobacterial MurI inhibits DNA gyrase activity, in addition to its precursor independent racemization function. The inhibition is not species-specific as E.coli gyrase is also inhibited but is enzyme-specific as topoisomerase I activity remains unaltered. The mechanism of inhibition is different from other well-known gyrase inhibitors. MurI binds to GyrA subunit of the enzyme leading to a decrease in DNA-binding of the holoenzyme. The sequestration of the gyrase by MurI results in inhibition of all reactions catalysed by DNA gyrase. MurI is thus not a typical potent inhibitor of DNA gyrase and instead its role could be in modulation of the gyrase activity.

Use of a codon alteration strategy in a novel approach to cloning the Mycobacterium tuberculosis diaminopimelic acid epimerase.

Previous attempts to express the diaminopimelate epimerase gene dapF of Mycobacterium tuberculosis in Escherichia coli resulted in undetectable enzyme yields. We used silent mutation of the first 10 codons of the recombinant ORF in an attempt to reduce the formation of secondary structures that might occur near the 5' end of the mRNA and inhibit translation. This significantly increased the yield of the enzyme, which was purified and characterized biochemically. This strategy could be generally applied to other mycobacterial genes that are difficult to express hetero-specifically and here provided pure M. tuberculosis DapF, a good foundation for future research in antimycobacterial agents.

Mammalian l-to-d-amino-acid-residue isomerase from platypus venom.

The presence of d-amino-acid-containing polypeptides, defensin-like peptide (DLP)-2 and Ornithorhyncus venom C-type natriuretic peptide (OvCNP)b, in platypus venom suggested the existence of a mammalian d-amino-acid-residue isomerase(s) responsible for the modification of the all-l-amino acid precursors. We show here that this enzyme(s) is present in the venom gland extract and is responsible for the creation of DLP-2 from DLP-4 and OvCNPb from OvCNPa. The isomerisation reaction is freely reversible and under well defined laboratory conditions catalyses the interconversion of the DLPs to full equilibration. The isomerase is approximately 50-60 kDa and is inhibited by methanol and the peptidase inhibitor amastatin. This is the first known l-to-d-amino-acid-residue isomerase in a mammal.