Hydantoin Racemase Research Articles

Identification of d-carbamoylase for biocatalytic cascade synthesis of d-tryptophan featuring high enantioselectivity

In this study, an enantioselective d-carbamoylase (AcHyuC) was identified from Arthrobacter crystallopoietes with optimum pH of 8.5, much more compatible with hydantoinase process than other reported d-N-carbamoylases. AcHyuC has a substrate preference for aromatic carbamoyl-compounds. The dynamic kinetic resolution (DKR) cascade was developed by combining this AcHyuC with hydantoin racemase from Arthrobacter aurescens (AaHyuA) and d-hydantoinase from Agrobacterium tumefaciens (AtHyuH) for enantioselective resolution of l-indolylmethylhydantoin into d-Trp. The optimum pH of DKR cascade reaction was determined to be 8.0, and PEG 400 could facilitate the reaction. As much as 80mM l-indolylmethylhydantoin could be fully converted to d-Trp within 12h at 0.5L scale, with 99.4% yield, >99.9% e.e. and productivity of 36.6gL−1d−1. This study provides a new d-carbamoylase compatible with the DKR cascade for efficient production of optically pure d-Trp from l-indolylmethylhydantoin.

L-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

Protein immobilization is proving to be an environmentally friendly strategy for manufacturing biochemicals at high yields and low production costs. This work describes the optimization of the so-called “double-racemase hydantoinase process,” a system of four enzymes used to produce optically pure l-amino acids from a racemic mixture of hydantoins. The four proteins were immobilized separately, and, based on their specific activity, the optimal whole relation was determined. The first enzyme, d,l-hydantoinase, preferably hydrolyzes d-hydantoins from d,l-hydantoins to N-carbamoyl-d-amino acids. The remaining l-hydantoins are racemized by the second enzyme, hydantoin racemase, and continue supplying substrate d-hydantoins to the first enzyme. N-carbamoyl-d-amino acid is racemized in turn to N-carbamoyl-l-amino acid by the third enzyme, carbamoyl racemase. Finally, the N-carbamoyl-l-amino acid is transformed to l-amino acid by the fourth enzyme, l-carbamoylase. Therefore, the product of one enzyme is the substrate of another. Perfect coordination of the four activities is necessary to avoid the accumulation of reaction intermediates and to achieve an adequate rate for commercial purposes. The system has shown a broad pH optimum of 7–9, with a maximum activity at 8 and an optimal temperature of 60 °C. Comparison of the immobilized system with the free protein system showed that the reaction velocity increased for the production of norvaline, norleucine, ABA, and homophenylalanine, while it decreased for l-valine and remained unchanged for l-methionine.

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

The “hydantoinase process” is a well-established method for the industrial production of optically pure d-amino acids. However, due to the strict d-enantioselectivity of most hydantoinase enzymes, the process is less efficient for l-amino acid production. We present a new chemo-enzymatic cascade reaction for natural and non-natural l-amino acid production from racemic mixtures of 5-monosubstituted hydantoins. This system comprised the following enzymes: d-hydantoinase from Agrobacterium tumefaciens BQL9, hydantoin racemase 1 from A. tumefaciens C58 and l-N-carbamoylase from Geobacillus stearothermophilus CECT43, together with N-succinyl-amino acid racemase from G. kaustophilus CECT4264. This latter presents catalytic promiscuity and racemizes N-carbamoyl-amino acids. This activity avoids the accumulation of N-carbamoyl-d-amino acid in the reaction due to the strict d-enantioselectivity of the hydantoinase. The optimum pH for the system proved to be 8.0, whereas optimum temperature range was 50–65°C, with the maximum reaction rate at 60°C. The metal ion cobalt was added directly to the reaction mixture (end concentration 1mM), but in the case of d-hydantoinase, overexpression in presence of 0.5mM Co2+ was also necessary. The enzymatic cascade reaction produced different optically pure l-amino acids by dynamic kinetic resolution, achieving 100% conversion even at high substrate concentrations (100mM) with no noticeable inhibition. This total conversion demonstrates that the “double-racemase hydantoinase process” upgrades the classical “hydantoinase process” for natural and non-natural l-amino acid production.

Characterization of hydantoin racemase predicted from the genome sequence ofLactobacillus pentosusKCA1

Background: Hydantoin racemase from Lactobacillus species of human origin has not been reported and characterized. The genome of Lactobacillus pentosus KCA1 has been sequenced and found to possess gene cassettes and open reading frames encoding the hydantoinase machinery, including a putative hydantoin racemase. Aims: To use bioinformatic tools to characterize the new hydantoin racemase predicted in the genome sequence of L. pentosus KCA1. Materials and Methods: Bioinformatic tools such as ClustalW algorithm was used to align hydantoin racemase from L. pentosus KCA1 with other hydantoin racemases extracted from the uniprot΢ database. I-TASSER was used for the prediction of secondary structure, 3-D model, similarity structure in PDB, and functional active binding site residues. Results: L. pentosus KCA1 hydantoin racemase showed significant amino acid sequence identity with hydantoin racemase from the selected bacterial organisms in the protein databank (PDB). The predicted secondary structure revealed 9 alpha-helices and 8 beta-strands. Functional prediction using enzyme partners predicted EC number 5.1.1.13 as the corresponding enzyme homolog (3eq5A) showing Cys83 and Cys187 as the potential active residues in KCA1 hydantoin racemase. The 3-D structure of KCA1 hydantoin racemase has a confidence score (C-score) of 1.2 that reflects a model of better quality, based on 3qvjA from PDB. Conclusion: The in silico data presented provides new insights into the potential activity and substrate specificity of hydantoin racemase from L. pentosus KCA1 and has proposed a mechanism for racemization of hydantoin derivatives that is consistent with the two-base process observed in other members of the Aspartate/Glutamate superfamily.

Crystallization and preliminary crystallographic studies of an active-site mutant hydantoin racemase fromSinorhizobium melilotiCECT4114

A recombinant active-site mutant of hydantoin racemase (C76A) from Sinorhizobium meliloti CECT 4114 (SmeHyuA) has been crystallized in the presence and absence of the substrate D,L-5-isopropyl hydantoin. Crystals of the SmeHyuA mutant suitable for data collection and structure determination were grown using the counter-diffusion method. X-ray data were collected to resolutions of 2.17 and 1.85 A for the free and bound enzymes, respectively. Both crystals belong to space group R3 and contain two molecules of SmeHyuA per asymmetric unit. The crystals of the free and complexed SmeHyuA have unit-cell parameters a = b = 85.43, c = 152.37 A and a = b = 85.69, c = 154.38 A, crystal volumes per protein weight (V(M)) of 1.94 and 1.98 A3 Da(-1) and solvent contents of 36.7 and 37.9%, respectively.

Recombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure d -Amino Acids

Two recombinant reaction systems for the production of optically pure D-amino acids from different D,L-5-monosubstituted hydantoins were constructed. Each system contained three enzymes, two of which were D-hydantoinase and D-carbamoylase from Agrobacterium tumefaciens BQL9. The third enzyme was hydantoin racemase 1 for the first system and hydantoin racemase 2 for the second system, both from A. tumefaciens C58. Each system was formed by using a recombinant Escherichia coli strain with one plasmid harboring three genes coexpressed with one promoter in a polycistronic structure. The D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest level of synthesis of the enzyme, thus avoiding intermediate accumulation, which decreases the reaction rate. Both systems were able to produce 100% conversion and 100% optically pure D-methionine, D-leucine, D-norleucine, D-norvaline, D-aminobutyric acid, D-valine, D-phenylalanine, D-tyrosine, and D-tryptophan from the corresponding hydantoin racemic mixture. For the production of almost all D-amino acids studied in this work, system 1 hydrolyzed the 5-monosubstituted hydantoins faster than system 2.

Site‐directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti

Hydantoin racemase enzyme plays a crucial role in the reaction cascade known as "hydantoinase process." In conjunction with a stereoselective hydantoinase and a stereospecific carbamoylase, it allows the total conversion from D,L-5-monosubstituted hydantoins, with a low rate of racemization, to optically pure D- or L-amino acids. Residues Cys76 and Cys181 belonging to hydantoin racemase from Sinorhizobium meliloti (SmeHyuA) have been proved to be involved in catalysis. Here, we report biophysical data of SmeHyuA Cys76 and Cys181 to alanine mutants, which point toward a two-base mechanism for the racemization of 5-monosubstituted hydantoins. The secondary and the tertiary structure of the mutants were not significantly affected, as shown by circular dichroism. Calorimetric and fluorescence experiments have shown that Cys76 is responsible for recognition and proton retrieval of D-isomers, while Cys181 is responsible for L-isomer recognition and racemization. This recognition process is further supported by measurements of protein stability followed by chemical denaturation in the presence of the corresponding compound.

Binding studies of hydantoin racemase from Sinorhizobium meliloti by calorimetric and fluorescence analysis

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d,l-5-monosubstituted hydantoins with a low velocity of racemization, to optically pure d-amino acids. Hydantoin racemase from Sinorhizobium meliloti was expressed in Escherichia coli. Calorimetric and fluorescence experiments were then carried out to obtain the thermodynamic binding parameters, Δ G, Δ H and Δ S for the inhibitors l- and d-5-methylthioethyl-hydantoin. The number of active sites is four per enzyme molecule (one per monomer), and the binding of the inhibitor is entropically and enthalpically favoured under the experimental conditions studied. In order to obtain information about amino acids involved in the active site, four different mutants were developed in which cysteines 76 and 181 were mutated to Alanine and Serine. Their behaviour shows that these cysteines are essential for enzyme activity, but only cysteine 76 affects the binding to these inhibitors.

D-Amino acid production by E. coli co-expressed three genes encoding hydantoin racemase, d-hydantoinase and N-carbamoyl- d-amino acid amidohydrolase

Three genes respectively encoding d-specific hydantoinase (DHHase), N-carbamoyl- d-amino acid amidohydrolase (DCHase) and hydantoin racemase (HRase) were co-expressed in E. coli in a system designed for the efficient enzymatic production of d-amino acids via a combination of hydantoin hydrolysis and hydantoin racemization. With the use of whole cells, the d-forms of eight amino acids – d-phenylalanine, d-tyrosine, d-tryptophan, O-benzyl- d-serine, d-valine, d-norvaline, d-leucine and d-norleucine – were efficiently converted from the corresponding dl-5-monosubtituted hydantoin compounds.

Purification and Characterization of Hydantoin Racemase fromMicrobacterium liquefaciensAJ 3912

A hydantoin racemase that catalyzed the racemization of 5-benzyl-hydantoin was detected in a cell-free extract of Microbacterium liquefaciens AJ 3912, a bacterial strain known to produce L-amino acids from their corresponding DL-5-substituted-hydantoins. This hydantoin racemase was purified 658-fold to electrophoretic homogeneity by serial chromatography. The N-terminal amino acid sequence of the enzyme showed homology with known hydantoin racemases from other microorganisms. The apparent molecular mass of the purified enzyme was 27 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 117 kDa on gel-filtration in the purification conditions, indicating a homotetrameric structure. The purified enzyme exhibited optimal activity at pH 8.2 and 55 degrees C, and showed a chiral preference for L-5-benzyl- rather than D-5-benzyl-hydantoin.

Evolutionary relationship and application of a superfamily of cyclic amidohydrolase enzymes

Cyclic amidohydrolases belong to a superfamily of enzymes that catalyze the hydrolysis of cyclic C-N bonds. They are commonly found in nucleotide metabolism of purine and pyrimidine. These enzymes share similar catalytic mechanisms and show considerable structural homologies, suggesting that they might have evolved from a common ancestral protein. Homology searches based on common mechanistic properties and three-dimensional protein structures provide clues to the evolutionary relationships of these enzymes. Among the superfamily of enzymes, hydantoinase has been highlighted by its potential for biotechnological applications in the production of unnatural amino acids. The enzymatic process for the production of optically pure amino acids consists of three enzyme steps: hydantoin racemase, hydantoinase, and N-carbamoylase. For efficient industrial application, some critical catalytic properties such as thermostability, catalytic activity, enantioselectivity, and substrate specificity require further improvement. To this end, isolation of new enzymes with desirable properties from natural sources and the optimization of enzymatic processes were attempted. A combination of directed evolution techniques and rational design approaches has made brilliant progress in the redesign of industrially important catalytic enzymes; this approach is likely to be widely applied to the creation of designer enzymes with desirable catalytic properties.

Molecular Cloning and Expression of thehyuGenes fromMicrobacterium liquefaciensAJ 3912, Responsible for the Conversion of 5-Substituted Hydantoins to α-Amino Acids, inEscherichia coli

A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to alpha-amino acids, was cloned in Escherichia coli and sequenced. Seven open reading frames (hyuP, hyuA, hyuH, hyuC, ORF1, ORF2, and ORF3) were identified on the 7.5 kb fragment. The deduced amino acid sequence encoded by the hyuA gene included the N-terminal amino acid sequence of the hydantoin racemase from M. liquefaciens AJ 3912. The hyuA, hyuH, and hyuC genes were heterologously expressed in E. coli; their presence corresponded with the detection of hydantoin racemase, hydantoinase, and N-carbamoyl alpha-amino acid amido hydrolase enzymatic activities respectively. The deduced amino acid sequences of hyuP were similar to those of the allantoin (5-ureido-hydantoin) permease from Saccharomyces cerevisiae, suggesting that hyuP protein might function as a hydantoin transporter.

Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58

A novel hydantoin racemase gene of Agrobacterium tumefaciens C58 ( AthyuA2) has been cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure and showed an apparent molecular mass of 27,000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100,000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 °C, respectively, with L-5-ethylhydantoin as substrate. Enzyme activity was strongly inhibited by Cu 2+ and Hg 2+. No effect on enzyme activity was detected with any other divalent cations, EDTA or DTT, suggesting that it is not a metalloenzyme. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.

Overexpression and characterization of hydantoin racemase from Agrobacterium tumefaciens C58

Hydantoin racemase enzyme together with a stereoselective hydantoinase and a stereospecific d-carbamoylase guarantee the total conversion from d, l-5-monosubstituted hydantoins with a low velocity of racemization to optically pure d-amino acids. In this work we have cloned and expressed the hydantoin racemase gene from two strains of Agrobacterium tumefaciens, C58 and LBA4404, in Escherichia coli BL21. The recombinant protein was purified in a one-step procedure by using immobilized cobalt affinity chromatography and showed an apparent molecular mass of 32,000 Da in SDS–gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of about 100,000 Da, suggesting that the native enzyme is a tetramer. The optimal conditions for hydantoin racemase activity were pH 7.5 and 55 °C with l-5-ethylhydantoin as substrate. Enzyme activity was slightly affected by the addition of Ni 2+ and Co 2+ and strongly inhibited by Cu 2+ and Hg 2+. No effect on enzyme activity was detected with Mn 2+, EDTA, or DTT. Kinetic studies showed the preference of the enzyme for hydantoins with short rather than long aliphatic side chains or hydantoins with aromatic rings.

Development of Dynamic Kinetic Resolution Processes for Biocatalytic Production of Natural and Nonnatural l-Amino Acids

Two different dynamic kinetic resolution processes for the production of a number of natural and nonnatural l-amino acids at 100% chemical and optical yield have recently been established at Degussa. The first process is based on the dynamic kinetic resolution of 5-monosubstituted hydantoins using tailor-made whole-cell biocatalysts coexpressing a l-carbamoylase, a hydantoin racemase, and a hydantoinase. The hydantoin-converting pathway was optimized by adjusting expression levels of the respective enzymes as well as by inverting the enantioselectivity of the d-selective hydantoinase. This resulted overall in a 50-fold improved productivity and significant reduction of biocatalyst cost. The second process is based on the dynamic kinetic resolution of N-acetyl amino acids using an acylase in combination with a novel racemase from Amycolatopsis orientalis subsp. lurida. This racemase could overcome the problem of substrate inhibition and requirement of high concentrations of divalent metal ions which limits...

Complete conversion of D,L-5-monosubstituted hydantoins with a low velocity of chemical racemization into D-amino acids using whole cells of recombinant Escherichia coli.

A reaction system was developed for the production of D-amino acids from D,L-5-monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the D-hydantoinase and D-carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli. The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed D-hydantoinase, D-carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic D,L-5-monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 degrees C), 100% conversion of D,L-5-(2-methylthioethyl)-hydantoin (15 mM) into D-methionine was obtained in 30 min.

Cloning and characterization of genes from Agrobacterium sp. IP I-671 involved in hydantoin degradation.

Cloning and sequencing of a 7.1 kb DNA fragment from Agrobacterium sp IP I-671 revealed seven open reading frames (ORFs) encoding D-hydantoinase, D-carbamoylase and putative hydantoin racemase, D-amino acid oxidase and NAD(P)H-flavin oxidoreductase. Two incomplete ORFs flanking the hydantoin utilization genes showed similarities to genes involved in transposition. Expression of the D-hydantoinase and D-carbamoylase gene in Escherichia coli gave mainly inactive protein concentrated in inclusion bodies, whereas homologous expression on an RSF1010 derivative increased hydantoinase and D-carbamoylase activity 2.5-fold and 10-fold, respectively, in this strain. Inactivation of the D-carbamoylase gene in Agrobacterium sp IP I-671 led to a complete loss of detectable carbamoylase activity whereas the low hydantoinase activity remaining after inactivation of the D-hydantoinase gene indicated the presence of a second hydantoinase-encoding gene. Two plasmids of 80 kb and 190 kb in size were identified by pulsed-field gel electrophoresis and the cloned hydantoin utilization genes were found to be localized on the 190 kb plasmid.

Organization of genes responsible for the stereospecific conversion of hydantoins to alpha-amino acids in Arthrobacter aurescens DSM 3747.

Arthrobacter aurescens DSM 3747 hydrolyzes stereospecifically 5'-monosubstituted hydantoins to alpha-amino acids. The genes involved in hydantoin utilization (hyu) were isolated on an 8.7-kb DNA fragment, and by DNA sequence analysis eight ORFs were identified. The hyu gene cluster includes four genes: hyuP encoding a putative transport protein, the hydantoin racemase gene hyuA, the hydantoinase gene hyuH, and the carbamoylase gene hyuC. The four genes are transcribed in the same direction. Upstream of hyuP and in opposite orientation to the hyu genes, three ORFs were found showing similarities to cytochrome P450 monooxygenase (ORF1, incomplete), to membrane proteins (ORF2), and to ferredoxin (ORF3). ORF8 was found downstream of hyuC and again in opposite orientation to the hyu genes. The gene product of ORF8 displayed similarities to the LacI/GalR family of transcriptional regulators. Reverse transcriptase PCR experiments and Northern blot analysis revealed that the genes hyuPAHC are coexpressed in A. aurescens after induction with 3-N-CH3-IMH. The expression of the hyu operon was not regulated by the putative regulator ORF8 as shown by gene disruption and mobility-shift experiments.

Development of an Escherichia coli whole cell biocatalyst for the production of l-amino acids

A whole cell biocatalyst for the enzymatic production of l-amino acids from hydantoins was created by coexpressing the genes encoding the l-hydantoinase, the l- N-carbamoylase and the hydantoin racemase from Arthrobacter aurescens in Escherichia coli. In order to construct a well balanced reaction system the enzymatic activity in the cells was varied by using vectors with different copy numbers for expression of the genes. Derivatives of pSC101, pACYC184 and pBR322 were employed for the various constructions and in one construct the hydantoinase gene was integrated into the E. coli chromosome. All constructs carried the E. coli rhamnose promoter system enabling gene expression control by transcriptional regulation. The productivity for l-tryptophan from the corresponding hydantoin was more than 6-fold higher than achieved with Arthrobacter aurescens.

Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization

In Arthrobacter aurescens DSM 3747 three enzymes are involved in the complete conversion of slowly racemizing 5′-monosubstituted d, l-hydantoins to l-amino acids, a stereoselective hydantoinase, a stereospecific l- N-carbamoylase and a hydantoin racemase. The gene encoding the hydantoin racemase, designated hyuA, was identified upstream of the previously described l- N-carbamoylase gene in the plasmid pAW16 containing genomic DNA of A. aurescens. The gene hyuA which encodes a polypeptide of 25.1 kDa, was expressed in Escherichia coli and the recombinant protein purified to homogeneity and further characterized. The optimal condition for racemase activity were pH 8.5 and 55°C with l-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCl 2 and iodoacetamide and stimulated by addition of dithiothreitol. No effect on enzyme activity was seen with EDTA. The enzyme showed preference for hydantoins with arylalkyl side chains. Kinetic studies revealed substrate inhibition towards the aliphatic substrate l-5-methylthioethylhydantoin. Enzymatic racemization of d-5-indolylmethylenehydantoin in D 2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization.