Enzymes
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- Name help_outline a D-5-monosubstituted hydantoin Identifier CHEBI:86339 Charge 0 Formula C3H3N2O2R SMILEShelp_outline [*][C@H]1NC(=O)NC1=O 2D coordinates Mol file for the small molecule Search links Involved in 3 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
- Name help_outline a L-5-monosubstituted hydantoin Identifier CHEBI:86340 Charge 0 Formula C3H3N2O2R SMILEShelp_outline [*][C@@H]1NC(=O)NC1=O 2D coordinates Mol file for the small molecule Search links Involved in 2 reaction(s) Find molecules that contain or resemble this structure Find proteins in UniProtKB for this molecule
Cross-references
RHEA:46624 | RHEA:46625 | RHEA:46626 | RHEA:46627 | |
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Reaction direction help_outline | undefined | left-to-right | right-to-left | bidirectional |
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Publications
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Hydantoinases and related enzymes as biocatalysts for the synthesis of unnatural chiral amino acids.
Altenbuchner J., Siemann-Herzberg M., Syldatk C.
A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time i ... >> More
A cascade of hydantoinase, N-carbamoylase and hydantoinracemase can be used for the production of natural and unnatural chiral D- and L-amino acids from chemically synthesized hydantoin derivatives. Potentially, 100% conversion and 100% optically pure amino acids can be obtained at the same time if racemic substrates are used. Recent research activities concentrate on newly isolated or improved enzymes and include directed evolution techniques, structure elucidation, studies of fusion proteins and the use of specially designed whole cell biocatalysts. << Less
Curr Opin Biotechnol 12:559-563(2001) [PubMed] [EuropePMC]
This publication is cited by 2 other entries.
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Molecular cloning, purification, and biochemical characterization of hydantoin racemase from the legume symbiont Sinorhizobium meliloti CECT 4114.
Martinez-Rodriguez S., Las Heras-Vazquez F.J., Mingorance-Cazorla L., Clemente-Jimenez J.M., Rodriguez-Vico F.
Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40 degrees C and 8.5, respectively. The enzyme showed a slight prefer ... >> More
Hydantoin racemase from Sinorhizobium meliloti was functionally expressed in Escherichia coli. The native form of the enzyme was a homotetramer with a molecular mass of 100 kDa. The optimum temperature and pH for the enzyme were 40 degrees C and 8.5, respectively. The enzyme showed a slight preference for hydantoins with short rather than long aliphatic side chains or those with aromatic rings. Substrates, which showed no detectable activity toward the enzyme, were found to exhibit competitive inhibition. << Less
Appl. Environ. Microbiol. 70:625-630(2004) [PubMed] [EuropePMC]
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Hydantoin racemase from Arthrobacter aurescens DSM 3747: heterologous expression, purification and characterization.
Wiese A., Pietzsch M., Syldatk C., Mattes R., Altenbuchner J.
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, de ... >> More
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 degrees C with L-5-benzylhydantoin as substrate. The enzyme was completely inhibited by HgCL2 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 D2O and NMR analysis showed that the hydrogen at the chiral center of the hydantoin is exchanged against solvent deuterium during the racemization. << Less
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Purification and characterization of the hydantoin racemase of Pseudomonas sp. strain NS671 expressed in Escherichia coli.
Watabe K., Ishikawa T., Mukohara Y., Nakamura H.
The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent mo ... >> More
The hydantoin racemase gene of Pseudomonas sp. strain NS671 had been cloned and expressed in Escherichia coli. Hydantoin racemase was purified from the cell extract of the E. coli strain by phenyl-Sepharose, DEAE-Sephacel, and Sephadex G-200 chromatographies. The purified enzyme had an apparent molecular mass of 32 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By gel filtration, a molecular mass of about 190 kDa was found, suggesting that the native enzyme is a hexamer. The optimal conditions for hydantoin racemase activity were pH 9.5 and a temperature of 45 degrees C. The enzyme activity was slightly stimulated by the addition of not only Mn2+ or Co2+ but also metal-chelating agents, indicating that the enzyme is not a metalloenzyme. On the other hand, Cu2+ and Zn2+ strongly inhibited the enzyme activity. Kinetic studies showed substrate inhibition, and the Vmax values for D- and L-5-(2-methylthioethyl)hydantoin were 35.2 and 79.0 mumol/min/mg of protein, respectively. The purified enzyme did not racemize 5-isopropylhydantoin, whereas the cells of E. coli expressing the enzyme are capable of racemizing it. After incubation of the purified enzyme with 5-isopropylhydantoin, the enzyme no longer showed 5-(2-methylthioethyl)hydantoin-racemizing activity. However, in the presence of 5-(2-methylthioethyl)hydantoin, the purified enzyme racemized 5-isopropylhydantoin completely, suggesting that 5-(2-methylthioethyl)hydantoin protects the enzyme from inactivation by 5-isopropylhydratoin. Thus, we examined the protective effect of various compounds and found that divalent-sulfur-containing compounds (R-S-R' and R-SH) have this protective effect. << Less
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Biochemical characterization of a novel hydantoin racemase from Agrobacterium tumefaciens C58.
Martinez-Rodriguez S., Las Heras-Vazquez F.J., Clemente-Jimenez J.M., Rodriguez-Vico F.
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 27000 Da in SDS-gel electrophoresis. Size exclusion chromatograph ... >> More
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 27000 Da in SDS-gel electrophoresis. Size exclusion chromatography analysis determined a molecular mass of approximately 100000 Da, suggesting that the native enzyme is a tetramer. The optimum pH and temperature for hydantoin racemase activity were 7.5 and 55 degrees 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. << Less
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Site-directed mutagenesis indicates an important role of cysteines 76 and 181 in the catalysis of hydantoin racemase from Sinorhizobium meliloti.
Martinez-Rodriguez S., Andujar-Sanchez M., Neira J.L., Clemente-Jimenez J.M., Jara-Perez V., Rodriguez-Vico F., Las Heras-Vazquez F.J.
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 op ... >> More
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. << Less
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Purification and characterization of hydantoin racemase from Microbacterium liquefaciens AJ 3912.
Suzuki S., Onishi N., Yokozeki K.
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-fo ... >> More
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. << Less
Biosci Biotechnol Biochem 69:530-536(2005) [PubMed] [EuropePMC]