Molecular Structure of Dihydroorotase:  A Paradigm for Catalysis through the Use of a Binuclear Metal Center<sup>,</sup>

Thoden, James B.; Phillips, George N.; Neal, Tamiko M.; Raushel, Frank M.; Holden, Hazel M.

doi:10.1021/bi010682i

Cited by 189 publications

(238 citation statements)

References 26 publications

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“…However, the zinc-zinc distance of 3.68 Å in HPRG is typical of dinuclear first transition row metal sites with bridging ligands like carboxylates or cysteine thiolate sulfur. Examples of dinuclear zinc sites can be seen in dihydroorotase (34), where two Zn 2ϩ ions, bridged by the carboxylate group of a carbamylated Lys residue, are at 3.48 Å distance. This also occurs in phosphotriesterase where two Zn 2ϩ ions are at 3.45 Å (35).…”

Section: Zinc-binding Site Of Skeletal Muscle Hprgmentioning

confidence: 99%

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

Mangani¹,

Meyer‐Klaucke²,

Moir³

et al. 2003

Journal of Biological Chemistry

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The AMP deaminase-associated variant of histidineproline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zincbinding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn 2؉ ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine. 11 cysteines of HPRG from different species are totally conserved, suggesting that five disulfide bridges are essential for the proper folding of the protein. At least another cysteine is present at different positions in the histidine-prolinerich domain of HPRG in all species, suggesting that this cysteine is the candidate for zinc ligation in the muscle variant of HPRG. The same conclusion is likely to be true for the six histidines used by the protein as zinc ligands. The presence in muscle HPRG of a specific zincbinding site permits us to envisage the addition of HPRG into the family of metallochaperones. In this view, HPRG may enhance the in vivo stability of metalloenzymes such as AMP deaminase. Histidine-proline-rich glycoprotein (HPRG)1 is an approximately 70-kDa glycoprotein that is present at a relatively high concentration in the plasma of vertebrates. Although the physiological role of this protein remains unclear, it has been implicated in a number of processes, including blood coagulation and fibrinolysis, immune response, and transport of metal ions (1). The cellular origin of the mouse protein has recently been defined by Northern blot analysis showing that the HPRG mRNA is localized specifically to the liver, suggesting that the previously described HPRG expression by immune cells is due to the acquisition of the plasma protein derived from the liver (2). In a previous paper we reported that denaturation of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.6) in acidic medium allows the chromatographic separation from the enzyme of a peptide with an amino acid composition significantly different from that derived from the available AMP deaminase cDNAs. N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of the novel protein to rabbit plasma HPRG although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension (3). We now report that the AMP deaminase-associated variant of HPRG can be isolated from rabbit skeletal muscle by a modification of the protocol used in our laboratory for the purification of AMP deaminase. The modification allows the partial dissociation of HPRG at a high degree of purity from the cellulose phosphate-bound enzyme.Rabbit plasma HPRG contains 53 histidine residues, of which 34 are located in the histidine-proline-rich d...

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Section: Zinc-binding Site Of Skeletal Muscle Hprgmentioning

confidence: 99%

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

Mangani¹,

Meyer‐Klaucke²,

Moir³

et al. 2003

Journal of Biological Chemistry

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“…An additional bridging ligand from the protein may include a carboxylated lysine from the end of β-strand 4, a glutamate from the end of β-strand three or four, or a cysteine from the end of β-strand 2 (8,9). Enzymes of this type include dihydroorotase (12), the phosphotriesterase homology protein (13), renal dipeptidase (14) and D-amino acid deacetylase (15). Many members of the amidohydrolase superfamily bind a single divalent cation, including cytosine (16) and adenosine deaminase (17).…”

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confidence: 99%

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Hall

Xiang

et al. 2007

Biochemistry

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NagA is a member of the amidohydrolase superfamily and catalyzes the deacetylation of N-acetyl-D-glucosamine-6-phosphate. The catalytic mechanism of this enzyme was addressed by the characterization of the catalytic properties of metal-substituted derivatives of NagA from Escherichia coli with a variety of substrate analogs. The reaction mechanism is of interest since NagA from bacterial sources is found with either one or two divalent metal ions in the active site. This observation indicates that there has been a divergence in the evolution of NagA and suggests that there are fundamental differences in the mechanistic details for substrate activation and hydrolysis. NagA from E. coli was inactivated by the removal of the zinc bound to the active site and the apo-enzyme reactivated upon incubation with one equivalent of Zn 2+ , Cd 2+ , Co 2+ , Mn 2+ , Ni 2+ or Fe 2+ . In the proposed catalytic mechanism the reaction is initiated by the polarization of the carbonyl group of the substrate via a direct interaction with the divalent metal ion and His-143. The invariant aspartate found at the end of β-strand 8 in all members of the amidohydrolase superfamily abstracts a proton from the metal-bound water molecule (or hydroxide) to promote the hydrolytic attack on the carbonyl group of the substrate. A tetrahedral intermediate is formed and then collapses with cleavage of the C-N bond after proton transfer to the leaving group amine by Asp-273. The lack of a solvent isotope effect by D 2 O and the absence of any changes to the kinetic constants with increases in solvent viscosity indicate that net product formation is not limited to any significant extent by proton transfer steps or the release of products. N-trifluoroacetyl-D-glucosamine-6-phosphate is hydrolyzed by NagA 26-fold faster than the corresponding N-acetyl derivative. This result is consistent with the formation or collapse of the tetrahedral intermediate as the rate limiting step in the catalytic mechanism of NagA.NagA 1 catalyzes the hydrolytic cleavage of N-acetyl-D-glucosamine-6-phosphate as illustrated in Scheme 1. The deacetylation of this compound provides a source of carbon and nitrogen by preparing this substrate for entry into the glycolytic pathway. This reaction is a key step in the catabolism of N-acetyl-D-glucosamine, derived from the degradation of chitin, and is an essential component for the biosynthesis of lipopolysaccharides and peptidoglycans. More recently, the reaction catalyzed by NagA has been shown to be an important step in the recycling of cell wall murein (1-3).The purification of NagA from Escherichia coli was originally reported by White and Pasternak (4). The enzyme oligomerizes as a tetramer and each subunit contains a reactive sulfhydryl group near the active site (5). In the forward reaction, inhibition occurs at high substrate † This work was supported in part by the NIH (GM 71790) and the Robert A. Welch Foundation (A-840). RSH was supported by a Chemical Biology Interface Training Grant (GM 08523). * To whom cor...

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“…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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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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Molecular Structure of Dihydroorotase: A Paradigm for Catalysis through the Use of a Binuclear Metal Center^,

Cited by 189 publications

References 26 publications

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

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Molecular Structure of Dihydroorotase: A Paradigm for Catalysis through the Use of a Binuclear Metal Center,

Cited by 189 publications

References 26 publications

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

Characterization of the Zinc-binding Site of the Histidine-Proline-rich Glycoprotein Associated with Rabbit Skeletal Muscle AMP Deaminase

N-Acetyl-d-glucosamine-6-phosphate Deacetylase: Substrate Activation via a Single Divalent Metal Ion

Metal Ion Dependence of RecombinantEscherichia coliAllantoinase

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Molecular Structure of Dihydroorotase: A Paradigm for Catalysis through the Use of a Binuclear Metal Center^,