Production of optically pure d- and l-α-amino acids by Bioconversion of d,l-5-monosubstituted hydantoin derivatives

Syldatk, Christoph; Läufer, Albrecht; Müller, Ralf; Höke, Hartmut

doi:10.1007/bfb0010231

Cited by 61 publications

(41 citation statements)

References 69 publications

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“…This concept was derived from the observation that the dihydropyrimidinase isolated from calf liver catalyzes not only dihydropyrimidines, but also the structurally related hydantoin derivatives (39). Based on this property, microbial D-hydantoinase has gained considerable attention for its ability to synthesize the less commonly occurring D-amino acids (34,44). E. coli HyuA, however, showed some novel properties in that its activity is absolutely dependent on metal ions and it has no activity on dihydropyrimidines.…”

Section: Discussionmentioning

confidence: 99%

Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

2000

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A superfamily of cyclic amidohydrolases, including dihydropyrimidinase, allantoinase, hydantoinase, and dihydroorotase, all of which are involved in the metabolism of purine and pyrimidine rings, was recently proposed based on the rigidly conserved structural domains in identical positions of the related enzymes. With these conserved domains, two putative cyclic amidohydrolase genes from Escherichia coli, flanked by related genes, were identified and characterized. From the genome sequence of E. coli, the allB gene and a putative open reading frame, tentatively designated as a hyuA (for hydantoin-utilizing enzyme) gene, were predicted to express hydrolases. In contrast to allB, high-level expression of hyuA in E. coli of a single protein was unsuccessful even under various induction conditions. We expressed HyuA as a maltose binding protein fusion protein and AllB in its native form and then purified each of them by conventional procedures. allB was found to encode a tetrameric allantoinase (453 amino acids) which specifically hydrolyzes the purine metabolite allantoin to allantoic acid. Another open reading frame, hyuA, located near 64.4 min on the physical map and known as a UUG start, coded for D-stereospecific phenylhydantoinase (465 amino acids) which is a homotetramer. As a novel enzyme belonging to a cyclic amidohydrolase superfamily, E. coli phenylhydantoinase exhibited a distinct activity toward the hydantoin derivative with an aromatic side chain at the 5 position but did not readily hydrolyze the simple cyclic ureides. The deduced amino acid sequence of the novel phenylhydantoinase shared a significant homology (>45%) with those of allantoinase and dihydropyrimidinase, but its functional role still remains to be elucidated. Despite the unclear physiological function of HyuA, its presence, along with the allantoin-utilizing AllB, strongly suggested that the cyclic ureides might be utilized as nutrient sources in E. coli.A superfamily of cyclic amidohydrolases (EC 3.5.2), including hydantoinase, dihydropyrimidinase, allantoinase, and dihydroorotase, has been recently proposed based on the functional and structural similarity of the related enzymes, providing evidence for an evolutionary common ancestor in related amidohydrolases (1,21,23). This family of enzymes is involved in the metabolism of pyrimidines and purines, sharing the property of hydrolyzing the cyclic amide bond of each substrate to the corresponding N-carbamyl amino acids (16,35). Dihydropyrimidinase and allantoinase catalyze the degradation of pyrimidines and purines, respectively, while hydantoinase, a microbial counterpart of dihydropyrimidinase, is also supposed to be involved in pyrimidine degradation. On the other hand, dihydroorotase participates in the de novo synthesis of pyrimidines (16). In a recent report, guanine deaminase from human kidney showed a common structural motif conserved in the family of cyclic amidohydrolases (46), expanding the family to more divergent enzymes acting on the purine and pyrimidine rings.Comparativ...

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Section: Discussionmentioning

confidence: 99%

Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

2000

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“…The stereoselective hydrolyses of DL-5-monosubstituted hydantoin compounds have been widely studied for the production of optically pure D-or L-amino acids (1,13), which are important intermediates for the production of various drugs and pharmaceuticals (2,26). Within certain species of bacteria, the hydantoin substrate compounds are first hydrolyzed by hydantoinase enzymes to form N-carbamoyl-␣-amino acids and then converted to corresponding free D-or L-amino acids catalyzed by N-carbamoyl-␣-amino acid amido hydrolase (Ncarbamoylase) enzymes (Fig.…”

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confidence: 99%

The Hydantoin Transport Protein from Microbacterium liquefaciens

Suzuki

Henderson

2006

J Bacteriol

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The gene hyuP from Microbacterium liquefaciens AJ 3912 with an added His 6 tag was cloned into the expression plasmid pTTQ18 in an Escherichia coli host strain. The transformed E. coli showed transport of radioisotope-labeled 5-substituted hydantoins with apparent K m values in the micromolar range. This activity exhibited a pH optimum of 6.6 and was inhibited by dinitrophenol, indicating the requirement of energy for the transport system. 5-Indolyl methyl hydantoin and 5-benzyl hydantoin were the preferred substrates, with selectivity for a hydrophobic substituent in position 5 of hydantoin and for the L isomer over the D isomer. Hydantoins with less hydrophobic substituents, cytosine, thiamine, uracil, allantoin, adenine, and guanine, were not effective ligands. The His-tagged hydantoin transport protein was located in the inner membrane fraction, from which it was solubilized and purified and its identity was authenticated.

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“…1,2) Stereoselective hydrolysis of D,L-5-monosubstituted hydantoin compounds is a widely used method of obtaining amino acids of high optical purity. 3,4) In the stereoselective method, hydantoin compounds, which can be chemically synthesized in many cases, 5) are used as the initial substrates.…”

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confidence: 99%

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

Suzuki

Takenaka

Onishi

et al. 2005

Bioscience, Biotechnology, and Biochemistry

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A DNA fragment from Microbacterium liquefaciens AJ 3912, containing the genes responsible for the conversion of 5-substituted-hydantoins to -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 -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.

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Production of optically pure d- and l-α-amino acids by Bioconversion of d,l-5-monosubstituted hydantoin derivatives

Cited by 61 publications

References 69 publications

Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

Functional Expression and Characterization of the Two Cyclic Amidohydrolase Enzymes, Allantoinase and a Novel Phenylhydantoinase, from Escherichia coli

The Hydantoin Transport Protein from Microbacterium liquefaciens

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

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