Crystal Structure of <scp>d</scp>-Hydantoinase from <i>Bacillus stearothermophilus</i>:  Insight into the Stereochemistry of Enantioselectivity<sup>,</sup>

Cheon, Young Hoon; Kim, Kwang Ho; Han, Kil Hwan; Abendroth, Jan; Niefind, Karsten; Schomburg, Dietmar; Wang, Jimin; Kim, Youngsoo

doi:10.1021/bi0201567

Cited by 90 publications

(110 citation statements)

References 30 publications

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“…Some, but not all, dinuclear hydrolase family members have their two metals bridged by a carbamylated lysine ligand (1,2,4,7,8,20,47). Significantly, we find that allantoinase sequences align with the active site regions of crystallized hydantoinases (1,2,8), dihydroorotase (47), and urease (20), as well as with a subgroup of hydantoinases that have not been structurally characterized (Fig. 2).…”

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confidence: 78%

“…Members of this family contain either mononuclear metal sites, such as the zinc site found in adenosine deaminase (56) and the iron site in cytosine deaminase (57), or dinuclear sites, such as the di-nickel site documented to occur in urease (20) and the di-zinc sites of dihydroorotase (47) and hydantoinase (1,2,8). Some, but not all, dinuclear hydrolase family members have their two metals bridged by a carbamylated lysine ligand (1,2,4,7,8,20,47). Significantly, we find that allantoinase sequences align with the active site regions of crystallized hydantoinases (1,2,8), dihydroorotase (47), and urease (20), as well as with a subgroup of hydantoinases that have not been structurally characterized (Fig.…”

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confidence: 99%

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Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Mulrooney

Hausinger

2003

J Bacteriol

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Allantoinase is a suspected dinuclear metalloenzyme that catalyzes the hydrolytic cleavage of the fivemember ring of allantoin (5-ureidohydantoin) to form allantoic acid. Recombinant Escherichia coli allantoinase purified from overproducing cultures amended with 2.5 mM zinc, 1 mM cobalt, or 1 mM nickel ions was found to possess ϳ1. Absorbance spectroscopy of the cobalt species reveals a band centered at 570 nm consistent with five-coordinate geometry. Dithiothreitol is a competitive inhibitor of the enzyme, with significant K i differences for the zinc and cobalt species (237 and 795 M, respectively). Circular dichroism spectroscopy revealed that the zinc enzyme utilizes only the S isomer of allantoin, whereas the cobalt allantoinase prefers the S isomer, but also hydrolyzes the R isomer at about 1/10 the rate. This is the first report for metal content of allantoinase from any source.Allantoinase (EC 3.5.2.5), present in a wide variety of bacteria, fungi, and plants, as well as a few animals, such as fish and amphibians, catalyzes the conversion of allantoin (5-ureidohydantoin) to allantoic acid by hydrolytic cleavage of the five-member hydantoin ring (33, 52). Allantoin is a key intermediate in nitrogen metabolism of leguminous plants, such as soybean (Fig. 1), where ureides provide the major transport form of symbiotically fixed dinitrogen (29,43,48,55). Until recently, it was thought that allantoin was formed directly from uric acid by the action of uric acid oxidase (39); however, it has now been established that the true product of soybean uric acid oxidase is 5-hydroxyisourate (22), which is then acted upon by the enzyme hydroxyisourate hydrolase to yield 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (42). This compound is then converted to allantoin, most likely by a yet unidentified enzyme. The product of allantoin hydrolysis, allantoate, is sequentially converted in soybean to ureidoglycine, ureidoglycolate, and glyoxylate with the ammonia released during these reactions utilized for plant cell growth. Variations of this pathway occur in other organisms, including Escherichia coli, which can grow on allantoin as the sole nitrogen source, but not as the sole carbon source. The E. coli allantoinase gene is part of a 12-gene regulon involved in allantoin degradation and nitrogen assimilation, and these genes are expressed only when the cells are grown under anaerobic conditions (10).On the basis of limited sequence similarities, allantoinase was suggested to belong to a broad family of metal-dependent amidohydrolases (17). Members of this family contain either mononuclear metal sites, such as the zinc site found in adenosine deaminase (56) and the iron site in cytosine deaminase (57), or dinuclear sites, such as the di-nickel site documented to occur in urease (20) and the di-zinc sites of dihydroorotase (47) and hydantoinase (1, 2, 8). Some, but not all, dinuclear hydrolase family members have their two metals bridged by a carbamylated lysine ligand (1,2,4,7,8,20,47). Significantly, we find that allantoi...

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confidence: 78%

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confidence: 99%

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Mulrooney

Hausinger

2003

J Bacteriol

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“…Recently, crystal structures of D-HYDs from both Thermus sp. (HYD Tsp ) (2) and B. stearothermophilus (HYD Bst ) (13) and of L-HYD from Arthrobacter aurescens (HYD Aau ) (1) have been solved. In addition, structures of several other amidohydrolases have been reported in the past years, including structures of the urease ␣-subunit from Klebsiella aerogenes (23) and Bacillus pasteurii (5,6), dihydroorotase from Escherichia coli (58), and phosphotriesterase from Pseudomonas diminuta (60).…”

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confidence: 99%

Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

Yun-qing

Yang

et al. 2003

J Bacteriol

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D-Hydantoinase (D-HYD) is an industrial enzyme that is widely used in the production of D-amino acids which are precursors for semisynthesis of antibiotics, peptides, and pesticides. This report describes the crystal structure of D-hydantoinase from Burkholderia pickettii (HYD Bp ) at a 2.7-Å resolution. The structure of HYD Bp consists of a core (␣/␤) 8 triose phosphate isomerase barrel fold and a ␤-sheet domain, and the catalytic active site consists of two metal ions and six highly conserved amino acid residues. Although HYD Bp shares only moderate sequence similarity with D-HYDs from Thermus sp. (HYD Tsp ) and Bacillus stearothermophilus (HYD Bst ), whose structures have recently been solved, the overall structure and the structure of the catalytic active site are strikingly similar. Nevertheless, the amino acids that compose the substrate-binding site are less conserved and have different properties, which might dictate the substrate specificity. Structural comparison has revealed insights into the molecular basis of the differential thermostability of D-HYDs. The more thermostable HYD Tsp contains more aromatic residues in the interior of the structure than HYD Bp and HYD Bst . Changes of large aromatic residues in HYD Tsp to smaller residues in HYD Bp or HYD Bst decrease the hydrophobicity and create cavities inside the structure. HYD Tsp has more salt bridges and hydrogen-bonding interactions and less oxidation susceptible Met and Cys residues on the protein surface than HYD Bp and HYD Bst . Besides, HYD Tsp also contains more rigid Pro residues. These factors are likely to make major contributions to the varying thermostability of these enzymes. This information could be exploited in helping to engineer more thermostable mesophilic enzymes.Dihydropyrimidinases (EC 3.5.2.2) are a family of enzymes that catalyze the reversible hydrolytic ring opening of the amide bond in five-or six-membered cyclic diamides (54, 62). In humans, they are responsible for the hydrolysis of dihydropyrimidines at the second step of reductive catabolism of pyrimidines. The homologous enzymes in microorganisms are hydantoinases (HYDs) which catalyze the hydrolysis of 5-substituted hydantoins to the enantiomerically pure Ncarbamyl amino acids. The latter can be converted chemically or enzymatically to the corresponding optically pure amino acids (50, 54, 64). In the biotechnology industry, HYD is widely used in the production of D-amino acids, including D-p-hydroxyphenylglycine, which are the precursors for semisynthesis of antibiotics, peptide hormones, pyrethroids, and pesticides (3,49,53,54). Depending on their stereoselectivities and specificities on substrates, HYDs are often classified as D-, L-, or non-enantio specific (47, 67).One major consideration in the application of enzymes for biocatalysis is thermostability. Screening of various thermophiles has successfully discovered several thermostable D-HYDs from Thermus sp. CBS30380 and Lu1220 (28), and Bacillus stearothermophilus SD-1 (38) and GH-2 (51). Substantial efforts...

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“…Second, the putative active site water molecule, which is likely to be present at the product acetate ACT1 O 1 position, lies significantly closer to the zinc ion at the ␣ 1 subsite (1.95 Å) than that at the ␤ site (2.84 Å). This is one striking difference between the active site here and those in other binuclear zinc hydrolases such as the ␣␤ members, where the water/hydroxide ion is almost symmetrically located between the two metal ions (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Therefore, the inhibitory metal ion at the ␣ site may hold the active site water tightly and thus perturb the stereochemical arrangements required for enzyme catalysis as it does in bovine carboxypeptidase A (38).…”

Section: Resultsmentioning

confidence: 89%

“…Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16,17).…”

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The Functional Role of the Binuclear Metal Center in d-Aminoacylase

Lai¹,

Chou²,

Ting³

et al. 2004

Journal of Biological Chemistry

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Our structural comparison of the TIM barrel metal-dependent hydrolase(-like) superfamily suggests a classification of their divergent active sites into four types: ␣␤-binuclear, ␣-mononuclear, ␤-mononuclear, and metal-independent subsets. The D-aminoacylase from Alcaligenes faecalis DA1 belongs to the ␤-mononuclear subset due to the fact that the catalytically essential , revealing that elimination of the ␤ site changes the coordination geometry of the ␣ ion with an enhanced affinity. Kinetic studies of the metal ligand mutants such as C96D indicate the uniqueness of the unusual bridging cysteine and its involvement in catalysis. Therefore, the two metal-binding sites in the Daminoacylase are interactive with partially mutual exclusion, thus resulting in widely different affinities for the activation/attenuation mechanism, in which the enzyme is activated by the metal ion at the ␤ site, but inhibited by the subsequent binding of the second ion at the ␣ site.D-Aminoacylase (EC 3.5.1.81) is an attractive candidate for production of D-amino acids through its catalysis of the deacetylation of N-acetyl-D-amino acids. Since D-amino acids are intermediates in the preparation of antibiotics, pesticides, and bioactive peptides, D-aminoacylase has commercial importance for the optical resolution of N-acetyl-DL-amino acids (1-2). The crystal structure of the 483-residue D-aminoacylase from Alcaligenes faecalis DA1 revealed that the enzyme is comprised of a small ␤ barrel and a catalytic TIM barrel with a unique 63-residue insertion (3). The structural similarity and the conserved metal ligands of four histidines and one aspartate suggest that D-aminoacylase belongs to the TIM barrel metal-dependent hydrolase superfamily. This superfamily includes a variety of enzymes with highly diverse substrates and has been coined an "evolutionary treasure" (4).To date, crystal structures of 14 members in the superfamily have been determined. As more structures are solved, more divergences in the metal centers and in the ␤-strand packing of the TIM barrel are observed (Fig. 1). Based on the binding site(s) of the catalytically essential metal ion(s), we classify these members into four types: ␣␤-binuclear (␣␤), ␣-mononuclear (␣), ␤-mononuclear (␤), and metal-independent subsets. Phosphotriesterase (homology protein), urease, dihydroorotase, renal dipeptidase, isoaspartyl didpeptidase, and dihydropyrimidinase comprise the ␣␤ subset (5-15). Murine adenosine deaminase and Escherichia coli cytosine deaminase belong to the ␣ subset (16, 17). D-Aminoacylase belongs to the ␤ subset due to the essential Zn 2ϩ binding tightly at the ␤ site, even though it possesses a weak ␣ binding site (3). In contrast, the activity assay and the crystal structure of uronate isomerase show that this enzyme does not display any specific metal requirement and thus belongs to the metal-independent subset (18,19).The functional requirement for the different metal centers is still unclear. Interestingly, Bacillus subtilis N-acetylglucosamine-6-phosphate deacetylase ...

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Crystal Structure of d-Hydantoinase from Bacillus stearothermophilus: Insight into the Stereochemistry of Enantioselectivity^,

Cited by 90 publications

References 30 publications

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

The Functional Role of the Binuclear Metal Center in d-Aminoacylase

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Crystal Structure of d-Hydantoinase from Bacillus stearothermophilus: Insight into the Stereochemistry of Enantioselectivity,

Cited by 90 publications

References 30 publications

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

The Functional Role of the Binuclear Metal Center in d-Aminoacylase

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Crystal Structure of d-Hydantoinase from Bacillus stearothermophilus: Insight into the Stereochemistry of Enantioselectivity^,