Amino Acid Racemase Research Articles

Diversity of D-Amino Acid Utilizing Bacteria From Kongsfjorden, Arctic and the Metabolic Pathways for Seven D-Amino Acids

D-amino acids (DAAs) are an important component of the refractory dissolved organic matter pool in the ocean. Microbes play a vital role in promoting the recycling of DAAs in the ocean. However, the diversity of marine DAA-utilizing bacteria and how they metabolize DAAs are seldom studied. Here, by enrichment culture with DAAs as the sole nitrogen source, bacteria of 12 families from three phyla were recovered from surface seawater and sediment from Kongsfjorden, Arctic, and seven DAA-utilizing bacterial strains were isolated. These strains have different DAA-utilizing abilities. Of the seven DAAs used, Halomonas titanicae SM1922 and Pseudoalteromonas neustonica SM1927 were able to utilize seven and five of them, respectively, while the other strains were able to utilize only one or two. Based on genomic, transcriptional and biochemical analyses, the key genes involved in DAA metabolism in each strain were identified and the metabolic pathways for the seven DAAs in these marine bacteria were identified. Conversion of DAAs into α-keto acids is generally the main pathway in marine DAA-utilizing bacteria, which is performed by several key enzymes, including DAA oxidoreductases/dehydrogenases, D-serine ammonia-lyases, D-serine ammonia-lyase DSD1s and DAA transaminases. In addition, conversion of DAAs into LAAs is another pathway, which is performed by amino acid racemases. Among the identified key enzymes, D-serine ammonia-lyase DSD1 and Asp racemase are first found to be employed by bacteria for DAA utilization. These results shed light on marine DAA-utilizing bacteria and the involved DAA metabolism pathways, offering a better understanding of the DAA recycling in the ocean.

In vivo directed enzyme evolution in nanoliter reactors with antimetabolite selection

Scoring changes in enzyme or pathway performance by their effect on growth behavior is a widely applied strategy for identifying improved biocatalysts. While in directed evolution this strategy is powerful in removing non-functional catalysts in selections, measuring subtle differences in growth behavior remains difficult at high throughput, as it is difficult to focus metabolic control on only one or a few enzymatic steps over the entire process of growth-based discrimination. Here, we demonstrate successful miniaturization of a growth-based directed enzyme evolution process. For cultivation of library clones we employed optically clear gel-like microcarriers of nanoliter volume (NLRs) as reaction vessels and used fluorescence-assisted particle sorting to estimate the growth behavior of each of the gel-embedded clones in a highly parallelized fashion. We demonstrate that the growth behavior correlates with the desired improvements in enzyme performance and that we can fine-tune selection stringency by including an antimetabolite in the assay. As a model enzyme reaction, we improve the racemization of ornithine, a possible starting block for the large-scale synthesis of sulphostin, by a broad-spectrum amino acid racemase and confirm the discriminatory power by showing that even moderately improved enzyme variants can be readily identified.

Distribution and evolution of the serine/aspartate racemase family in plants

Previous studies have shown that several d-amino acids are widely present in plants, and serine racemase (SerR), which synthesizes d-serine in vivo, has already been identified from three plant species. However, the full picture of the d-amino acid synthesis pathway in plants is not well understood. To clarify the distribution of amino acid racemases in plants, we have cloned, expressed and characterized eight SerR homologous genes from five plant species, including green alga. These SerR homologs exhibited racemase activity towards serine or aspartate and were identified on the basis of their maximum activity as SerR or aspartate racemase (AspR). The plant AspR gene is identified for the first time from Medicago truncatula, Manihot esculenta, Solanum lycopersicum, Sphagnum girgensohnii and Spirogyra pratensis. In addition to the AspR gene, three SerR genes are identified in the former three species. Phylogenetic tree analysis showed that SerR and AspR are widely distributed in plants and form a serine/aspartate racemase family cluster. The catalytic efficiency (kcat/Km) of plant AspRs was more than 100 times higher than that of plant SerRs, suggesting that d-aspartate, as well as d-serine, can be synthesized in vivo by AspR. The amino acid sequence alignment and comparison of the chromosomal gene arrangement have revealed that plant AspR genes independently evolved from SerR in each ancestral lineage of plant species by gene duplication and acquisition of two serine residues at position 150 to 152.

Structural insights into the catalysis and substrate specificity of cyanobacterial aspartate racemase McyF

L-amino acids represent the most common amino acid form, most notably as protein residues, whereas D-amino acids, despite their rare occurrence, play significant roles in many biological processes. Amino acid racemases are enzymes that catalyze the interconversion of L- and/or D-amino acids. McyF is a pyridoxal 5′-phosphate (PLP) independent amino acid racemase that produces the substrate D-aspartate for the biosynthesis of microcystin in the cyanobacterium Microcystis aeruginosa PCC7806. Here we report the crystal structures of McyF in complex with citrate, L-Asp and D-Asp at 2.35, 2.63 and 2.80 Å, respectively. Structural analyses indicate that McyF and homologs possess highly conserved residues involved in substrate binding and catalysis. In addition, residues Cys87 and Cys195 were clearly assigned to the key catalytic residues of “two bases” that deprotonate D-Asp and L-Asp in a reaction independent of PLP. Further site-directed mutagenesis combined with enzymatic assays revealed that Glu197 also participates in the catalytic reaction. In addition, activity assays proved that McyF could also catalyze the interconversion of L-MeAsp between D-MeAsp, the precursor of another microcystin isoform. These findings provide structural insights into the catalytic mechanism of aspartate racemase and microcystin biosynthesis.

Catalytic mechanism and properties of pyridoxal 5'-phosphate independent racemases: how enzymes alter mismatched acidity and basicity.

Covering: up to March 2019 Amino acid racemases and epimerases are key enzymes that invert the configuration of common amino acids and supply many corresponding d-isomers in living organisms. Some d-amino acids are inherently bioactive, whereas others are building blocks for important biomolecules, for example lipid II, the bacterial cell wall precursor. Peptides containing them have enhanced proteolytic stability and can act as important recognition elements in mammalian systems. Selective inhibition of certain amino acid racemases (e.g. glutamate racemase) is believed to offer a promising target for new antibacterial drugs effective against pathogens resistant to current antibiotics. Many amino acid racemases employ imine formation with pyridoxal phosphate (PLP) as a cofactor to accelerate the abstraction of the alpha proton. However, the group reviewed herein achieves racemization of free amino acids without the use of cofactors or metals, and uses a thiol/thiolate pair for deprotonation and reprotonation. All bacteria and higher plants contain such enzymes, for example diaminopimelate epimerase, which is required for lysine biosynthesis in these organisms. This process cannot be accomplished without an enzyme catalyst as the acidities of a thiol and the substrate α-hydrogen are inherently mismatched by at least 10 orders of magnitude. This review describes the structural and mechanistic studies on PLP-independent racemases and the evolving view of key enzymatic machinery that accomplishes these remarkable transformations.

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.

Production of d-Branched-Chain Amino Acids by Lactic Acid Bacteria Carrying Homologs to Isoleucine 2-Epimerase of Lactobacillus buchneri.

Isoleucine 2-epimerase (ILEP) is a novel branched-chain amino acid racemase isolated from Lactobacillus buchneri. In this study, we examined production of free d-branched-chain amino acids such as d-valine, d-leucine, and d-allo-isoleucine, using lactic acid bacteria carrying homologs to ILEP. Twelve selected strains of lactic acid bacteria were grown at optimal growth temperatures and accumulation of d-branched-chain amino acids in the medium was monitored in exponential, early stationary, and stationary phases. To analyze the d-branched-chain amino acids, enantiomers in the medium were initially converted into diastereomers using pre-column derivatization with o-phthaldialdehyde plus N-isobutyryl-l-cysteine. The resultant fluorescent isoindole derivatives were analyzed on an octadecylsilyl stationary phase using ultra-high performance liquid chromatography. The analyses revealed that the seven following lactic acid bacteria carrying homologs showing 53–60% amino acid sequence identity to the L. buchneri ILEP accumulate d-branched-chain amino acids: Lactobacillus fermentum and Weissella paramesenteroides produce d-valine, d-leucine, and d-allo-isoleucine; Lactobacillus reuteri, Leuconostoc mesenteroides subsp. mesenteroides, and Leuconostoc gelidum subsp. gasicomitatum accumulate d-leucine and d-allo-isoleucine; and Lactobacillus vaginalis and Leuconostoc pseudomesenteroides produce d-allo-isoleucine. These results suggest that d-branched-chain amino acids are produced by a variety of lactic acid bacteria species, particularly those carrying homologs to the ILEP.

A Novel PLP-Dependent Alanine/Serine Racemase From the Hyperthermophilic Archaeon Pyrococcus horikoshii OT-3.

We recently identified and characterized a novel broad substrate specificity amino acid racemase (BAR) from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Three genes, PH0782, PH1423, and PH1501, encoding homologs exhibiting about 45% sequence identity with BAR were present in the P. horikoshii genome. In this study, we detected pyridoxal 5′-phosphate (PLP)-dependent amino acid racemase activity in the protein encoded by PH0782. The enzyme showed activity toward Ala, Ser, Thr, and Val, but the catalytic efficiency with Thr or Val was much lower than with Ala or Ser. The enzyme was therefore designated Ala/Ser racemase (ASR). Like BAR, ASR was highly stable at high temperatures and over a wide range of pHs, though its hexameric structure differed from the dimeric structure of BAR. No activity was detected in K291A or D234A ASR mutants. This suggests that, as in Ile 2-epimerase (ILEP) from Lactobacillus buchneri JCM1115, these residues are involved in Schiff base formation and substrate interaction, respectively. Unlike BAR, enhanced ASR activity was not detected in P. horikoshii cells cultivated in the presence of D-Ala or D-Ser. This is the first description of a PLP-dependent fold type I ASR in archaea.

A Broad Spectrum Racemase in Pseudomonas putida KT2440 Plays a Key Role in Amino Acid Catabolism.

The broad-spectrum amino acid racemase (Alr) of Pseudomonas putida KT2440 preferentially interconverts the l- and d-stereoisomers of Lys and Arg. Despite conservation of broad-spectrum racemases among bacteria, little is known regarding their physiological role. Here we explore potential functional roles for Alr in P. putida KT2440. We demonstrate through cellular fractionation that Alr enzymatic activity is found in the periplasm, consistent with its putative periplasm targeting sequence. Specific activity of Alr is highest during exponential growth, and this activity corresponds with an increased accumulation of d-Lys in the growth medium. An alr gene knockout strain (Δalr) was generated and used to assess potential roles for the alr gene in peptidoglycan structure, producing soluble signaling compounds, and amino acid metabolism. The stationary phase peptidoglycan structure did not differ between wild-type and Δalr strains, indicating that products resulting from Alr activity are not incorporated into peptidoglycan under these conditions. RNA-seq was used to assess differences in the transcriptome between the wild-type and Δalr strains. Genes undergoing differential expression were limited to those involved in amino acid metabolism. The Δalr strain exhibited a limited capacity for catabolism of l-Lys and l-Arg as the sole source of carbon and nitrogen. This is consistent with a predicted role for Alr in catabolism of l-Lys by virtue of its ability to convert l-Lys to d-Lys, which is further catabolized through the l-pipecolate pathway. The metabolic profiles here also implicate Alr in catabolism of l-Arg, although the pathway by which d-Arg is further catabolized is not clear at this time. Overall, data presented here describe the primary role of Alr as important for basic amino acid metabolism.

One‐step Synthesis of α‐Keto Acids from Racemic Amino Acids by A Versatile Immobilized Multienzyme Cell‐free System

AbstractThe elevated value of α‐keto acids has pushed scientists to explore more efficient and less expensive alternatives for their synthesis. In this work, an immobilized tri‐enzyme system that produced α‐keto acids in “one‐pot” from l‐ or racemic mixtures of diverse amino acids was presented. The system combined a broad‐spectrum amino acid racemase (BsrV), a d‐amino acid oxidase (DAAO) and catalase (CAT). BsrV racemized l‐amino acids into their d‐enantiomers, DAAO catalyzed the stereospecific oxidative deamination of the d‐amino acids into their corresponding α‐keto acids, ammonium ion, and H2O2. Finally, CAT converted the inactivating H2O2 into H2O and O2, which can be reused by the oxidase reaction. BsrV thermal stability was improved 3,300‐fold by immobilizing the enzyme on glyoxyl‐activated agarose beads. DAAO and CAT were co‐immobilized on agarose beads functionalized with glutaraldehyde groups for enhancing their stabilities and eliminating H2O2 in a much more effective way. To show the versatility of this system, racemic mixtures of amino acids were converted in their corresponding α‐keto acids. The coupling of the three immobilized enzymes permitted conversions of approximately 99 % through a dynamic kinetic resolution process. This system conserved 100 % of its initial effectiveness after 8 reaction cycles. Collectively, our innovative tri‐enzyme system for the synthesis of α‐keto acids opens the door for a cheapening in the production of many pharmaceutical and cosmetics.

Cystathionine β-lyase is involved in d-amino acid metabolism.

Non-canonical d-amino acids play important roles in bacteria including control of peptidoglycan metabolism and biofilm disassembly. Bacteria appear to produce non-canonical d-amino acids to adapt to various environmental changes, and understanding the biosynthetic pathways is important. We identified novel amino acid racemases possessing the ability to produce non-canonical d-amino acids in Escherichia coli and Bacillus subtilis in our previous study, whereas the biosynthetic pathways of these d-amino acids still remain unclear. In the present study, we demonstrated that two cystathionine β-lyases (MetC and MalY) from E. coli produce non-canonical d-amino acids including non-proteinogenic amino acids. Furthermore, MetC displayed d- and l-serine (Ser) dehydratase activity. We characterised amino acid racemase, Ser dehydratase and cysteine lyase activities, and all were higher for MetC. Interestingly, all three activities were at a comparable level for MetC, although optimal conditions for each reaction were distinct. These results indicate that MetC and MalY are multifunctional enzymes involved in l-methionine metabolism and the production of d-amino acids, as well as d- and l-Ser metabolism. To our knowledge, this is the first evidence that cystathionine β-lyase is a multifunctional enzyme with three different activities.

A Novel Bifunctional Amino Acid Racemase With Multiple Substrate Specificity, MalY From Lactobacillus sakei LT-13: Genome-Based Identification and Enzymological Characterization.

The Lactobacillus sakei strain LK-145 isolated from Moto, a starter of sake, produces potentially large amounts of three D-amino acids, D-Ala, D-Glu, and D-Asp, in a medium containing amylase-digested rice as a carbon source. The comparison of metabolic pathways deduced from the complete genome sequence of strain LK-145 to the type culture strain of Lactobacillus sakei strain LT-13 showed that the L- and D-amino acid metabolic pathways are similar between the two strains. However, a marked difference was observed in the putative cysteine/methionine metabolic pathways of strain LK-145 and LT-13. The cystathionine β-lyase homolog gene malY was annotated only in the genome of strain LT-13. Cystathionine β-lyase is an important enzyme in the cysteine/methionine metabolic pathway that catalyzes the conversion of L-cystathionine into L-homocysteine. In addition to malY, most genome-sequenced strains of L. sakei including LT-13 lacked the homologous genes encoding other putative enzymes in this pathway. Accordingly, the cysteine/methionine metabolic pathway likely does not function well in almost all strains of L. sakei. We succeeded in cloning and expressing the malY gene from strain LT-13 (Ls-malY) in the cells of Escherichia coli BL21 (DE3) and characterized the enzymological properties of Ls-MalY. Spectral analysis of purified Ls-MalY showed that Ls-MalY contained a pyridoxal 5′-phosphate (PLP) as a cofactor, and this observation agreed well with the prediction based on its primary structure. Ls-MalY showed amino acid racemase activity and cystathionine β-lyase activity. Ls-MalY showed amino acid racemase activities in various amino acids, such as Ala, Arg, Asn, Glu, Gln, His, Leu, Lys, Met, Ser, Thr, Trp, and Val. Mutational analysis revealed that the 𝜀-amino group of Lys233 in the primary structure of Ls-MalY likely bound to PLP, and Lys233 was an essential residue for Ls-MalY to catalyze both the amino acid racemase and β-lyase reactions. In addition, Tyr123 was a catalytic residue in the amino acid racemase reaction but strongly affected β-lyase activity. These results showed that Ls-MalY is a novel bifunctional amino acid racemase with multiple substrate specificity; both the amino acid racemase and β-lyase reactions of Ls-MalY were catalyzed at the same active site.

Broad-specificity amino acid racemase, a novel non-antibiotic selectable marker for transgenic plants

The broad-specificity amino acid racemase (Bsar) from Pseudomonas putida catalyzes the racemization of various amino acids, offering a flexible and feasible platform to develop a new non-antibiotic selectable marker system for plant transformation. In the present study, we demonstrated that a Bsar variant, Bsar-R174K, that is useful as a selectable marker gene in Arabidopsis and rice that were susceptible to l-lysine and D-alanine. The introduction of wild-type Bsar, Bsar-R174K or Bsar-R174A into E. coli lysine or asparagine auxotrophs was able to rescue the growth of these microorganisms in minimal media supplemented with selectable amino acid enantiomers. The transformation of Arabidopsis with Bsar or Bsar variants based on d-alanine selection revealed that Bsar-R174K had the greatest efficiency (2.40%), superior to kanamycin selection-based transformation (1.10%). Whereas, l-lysine-based selection exhibited lower efficiency for Bsar-R174K (0.17%). The progenies of selected Bsar-R174K transgenic Arabidopsis revealed normal growth properties. In addition, Bsar-R174K transgenic rice was obtained on l-lysine medium with an efficiency of 0.9%, and the progenies of the transgenic rice revealed morphologically normal phenotypes comparable with their wild-type counterparts. This study presents the first report of broad range amino acid racemase Bsar-R174K as a non-antibiotic selectable marker system applied in transgenic plants.

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, 100mM 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.

Genome Sequence of Lactobacillus sakei LK-145 Isolated from a Japanese Sake Cellar as a High Producer of d-Amino Acids.

ABSTRACTThis announcement reports the complete genome sequence of strain LK-145 of Lactobacillus sakei isolated from a Japanese sake cellar as a potent strain for the production of large amounts of d-amino acids. Three putative genes encoding an amino acid racemase were identified.

An overview on D-amino acids.

More than half a century ago researchers thought that D-amino acids had a minor function compared to L-enantiomers in biological processes. Many evidences have shown that D-amino acids are present in high concentration in microorganisms, plants, mammals and humans and fulfil specific biological functions. In the brain of mammals, D-serine (D-Ser) acts as a co-agonist of the N-methyl-D-aspartate (NMDA)-type glutamate receptors, responsible for learning, memory and behaviour. D-Ser metabolism is relevant for disorders associated with an altered function of the NMDA receptor, such as schizophrenia, ischemia, epilepsy and neurodegenerative disorders. On the other hand, D-aspartate (D-Asp) is one of the major regulators of adult neurogenesis and plays an important role in the development of endocrine function. D-Asp is present in the neuroendocrine and endocrine tissues and testes, and regulates the synthesis and secretion of hormones and spermatogenesis. Also food proteins contain D-amino acids that are naturally originated or processing-induced under conditions such as high temperatures, acid and alkali treatments and fermentation processes. The presence of D-amino acids in dairy products denotes thermal and alkaline treatments and microbial contamination. Two enzymes are involved in the metabolism of D-amino acids: amino acid racemase in the synthesis and D-amino acid oxidase in the degradation.

Crystal structure of the novel amino-acid racemase isoleucine 2-epimerase from Lactobacillus buchneri.

Crystal structures of Lactobacillus buchneri isoleucine 2-epimerase, a novel branched-chain amino-acid racemase, were determined for the enzyme in the apo form, in complex with pyridoxal 5'-phosphate (PLP), in complex with N-(5'-phosphopyridoxyl)-L-isoleucine (PLP-L-Ile) and in complex with N-(5'-phosphopyridoxyl)-D-allo-isoleucine (PLP-D-allo-Ile) at resolutions of 2.77, 1.94, 2.65 and 2.12 Å, respectively. The enzyme assembled as a tetramer, with each subunit being composed of N-terminal, C-terminal and large PLP-binding domains. The active-site cavity in the apo structure was much more solvent-accessible than that in the PLP-bound structure. This indicates that a marked structural change occurs around the active site upon binding of PLP that provides a solvent-inaccessible environment for the enzymatic reaction. The main-chain coordinates of the L. buchneri isoleucine 2-epimerase monomer showed a notable similarity to those of α-amino-ℇ-caprolactam racemase from Achromobactor obae and γ-aminobutyrate aminotransferase from Escherichia coli. However, the amino-acid residues involved in substrate binding in those two enzymes are only partially conserved in L. buchneri isoleucine 2-epimerase, which may account for the differences in substrate recognition by the three enzymes. The structures bound with reaction-intermediate analogues (PLP-L-Ile and PLP-D-allo-Ile) and site-directed mutagenesis suggest that L-isoleucine epimerization proceeds through abstraction of the α-hydrogen of the substrate by Lys280, while Asp222 serves as the catalytic residue adding an α-hydrogen to the quinonoid intermediate to form D-allo-isoleucine.

First characterization of an archaeal amino acid racemase with broad substrate specificity from the hyperthermophile Pyrococcus horikoshii OT-3

A novel amino acid racemase with broad substrate specificity (BAR) was recently isolated from the hyperthermophilic archaeon Pyrococcus horikoshii OT-3. Characterization of this enzyme has been difficult, however, because the recombinant enzyme is produced mainly as an inclusion body in Escherichia coli. In this study, expression of the recombinant protein into the soluble fraction was markedly improved by co-expression with chaperone molecules. The purified enzyme retained its full activity after incubation at 80°C for at least 2h in buffer (pH 7-10), making this enzyme the most thermostable amino acid racemase so far known. Besides the nine amino acids containing hydrophobic and aromatic amino acids previously reported (Kawakami etal., Amino Acids, 47, 1579-1587, 2015), the enzyme exhibited substantial activity toward Thr (about 42% of relative activity toward Phe) and showed no activity toward Arg, His, Gln, and Asn. The substrate specificity of this enzyme thus differs markedly from those of other known amino acid racemases. In particular, the high reaction rate with Trp and Tyr, in addition to Leu, Met and Phe as substrates is a noteworthy feature of this enzyme. The high reactivity toward Trp and Tyr, as well as extremely high thermostability, is likely a major advantage of using BAR for biochemical conversion of these aromatic amino acids.

Exploiting racemases.

Chiral resolutions of racemic mixtures are limited to a theoretical yield of 50%. This yield can be doubled by integration of a step-wise or continuous racemization of the non-desired enantiomer. Many of the different routes along which the racemization step can be conducted require harsh treatments and are therefore often incompatible with the highly functionalized state of many compounds relevant for the life science industries. Employing enzymatic catalysis for racemization can therefore be highly beneficial. Racemases allow racemization in one reaction step. Most representatives from this group are found in the domain of amino acid or amino acid derivative racemization, with few other examples, notably the racemization of mandelic acid. Corresponding to the importance of enantiospecific conversion of amino acid precursor racemates for the production of enantiopure amino acids, the most important biotechnological use for racemases is the racemization of such precursors. However, alternative uses, in particular for mandelate and amino acid racemases, are emerging. Here, we summarize the natural roles of racemases and their occurrence, the applications, and the biochemistry and engineering of this promising class of biocatalysts.