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Richard I. Christopherson - One of the best experts on this subject based on the ideXlab platform.
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Structures of Ligand-free and Inhibitor Complexes of Dihydroorotase from Escherichia coli: Implications for Loop Movement in Inhibitor Design
Journal of molecular biology, 2007Co-Authors: Mihwa Lee, Richard I. Christopherson, Camilla Chan, J Mitchell Guss, Stephen C. Graham, Megan J MaherAbstract:Dihydroorotase (DHOase) catalyzes the reversible cyclization of N-carbamyl-L-aspartate (CA-asp) to L-dihydroorotate (DHO) in the de novo biosynthesis of pyrimidine nucleotides. DHOase is a potential anti-malarial drug target as malarial parasites can only synthesize pyrimidines via the de novo pathway and do not possess a salvage pathway. Here we report the structures of Escherichia coli DHOase crystallized without ligand (1.7 A resolution) and in the presence of the inhibitors 2-oxo-1,2,3,6-tetrahydropyrimidine-4,6-dicarboxylate (HDDP; 2.0 A) and 5-fluoroorotate (FOA, 2.2 A). These are the first crystal structures of DHOase-inhibitor complexes, providing structural information on the mode of inhibitor binding. HDDP possesses features of both the substrate and product, and ligates the Zn atoms in the active site. In addition, HDDP forms hydrogen bonds to the flexible loop (residues 105-115) stabilizing the "loop-in" conformation of the flexible loop normally associated with the presence of CA-asp in the active site. By contrast, FOA, a product-like inhibitor, binds to the active site in a similar fashion to DHO but does not ligate the Zn atoms directly nor stabilize the loop-in conformation. These structures define the necessary features for the future design of improved inhibitors of DHOase.
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13C and 15N isotope effects for conversion of L-dihydroorotate to N-carbamyl-L-aspartate using Dihydroorotase from hamster and Bacillus caldolyticus.
Biochemistry, 2006Co-Authors: Mark Anderson, Danny T. C. Huang, W. Wallace Cleland, Camilla Chan, Maryam Shojaei, Richard I. ChristophersonAbstract:In the pyrimidine biosynthetic pathway, N-carbamyl-l-aspartate (CA-asp) is converted to l-dihydroorotate (DHO) by Dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 °C for hamster DHOase; 37, 50, and 60 °C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the β-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary i...
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Dihydroorotase from Escherichia coli: Loop Movement and Cooperativity between Subunits
Journal of molecular biology, 2005Co-Authors: Mihwa Lee, Richard I. Christopherson, Camilla Chan, J.m. Guss, Megan J MaherAbstract:Escherichia coli Dihydroorotase has been crystallized in the presence of the product, l -dihydroorotate ( l -DHO), and the structure refined at 1.9 A resolution. The structure confirms that previously reported (PDB entry 1J79), crystallized in the presence of the substrate N-carbamyl- d , l -aspartate ( d , l -CA-asp), which had a dimer in the asymmetric unit, with one subunit having the substrate, l -CA-asp bound at the active site and the other having l -DHO. Importantly, no explanation for the unusual structure was given. Our results now show that a loop comprised of residues 105–115 has different conformations in the two subunits. In the case of the l -CA-asp-bound subunit, this loop reaches in toward the active site and makes hydrogen-bonding contact with the bound substrate molecule. For the l -DHO-bound subunit, the loop faces in the opposite direction and forms part of the surface of the protein. Analysis of the kinetics for conversion of l -DHO to l -CA-asp at low concentrations of l -DHO shows positive cooperativity with a Hill coefficient n=1.57(±0.13). Communication between subunits in the dimer may occur via cooperative conformational changes of the side-chains of a tripeptide from each subunit: Arg256-His257-Arg258, near the subunit interface.
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Crystallization of hamster Dihydroorotase: involvement of a disulfide‐linked tetrameric form
Acta Crystallographica Section D Biological Crystallography, 2003Co-Authors: Megan J Maher, J Mitchell Guss, Charles A Collyer, D.t.c. Huang, Richard I. ChristophersonAbstract:Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway. The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD. The hamster DHOase domain has been cloned and expressed in Escherichia coli. Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature. Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer. This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer. The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees. Data have been recorded to 4.3 and 4.0 A resolution, respectively.
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Crystallization of hamster Dihydroorotase: involvement of a disulfide-linked tetrameric form.
Acta crystallographica. Section D Biological crystallography, 2003Co-Authors: Megan J Maher, Danny T. C. Huang, J Mitchell Guss, Charles A Collyer, Richard I. ChristophersonAbstract:Dihydroorotase (DHOase) catalyses the formation of L-dihydroorotate (DHO) in the de novo pyrimidine biosynthetic pathway. The type I DHOase domain from hamster forms part of the trifunctional enzyme CAD. The hamster DHOase domain has been cloned and expressed in Escherichia coli. Solutions of the homodimeric protein convert to a homotetrameric species when incubated at ambient temperature. Formation of the tetrameric species is mediated via disulfide linkages between single free cysteine residues on the surface of each monomer. This process is also observed under conditions used for crystallization of the hamster DHOase domain; crystals composed exclusively of the tetrameric species grow from solutions containing as little as 10% tetramer. The crystallization of pure tetrameric DHOase results in two crystal forms: form I, with space group C222(1) and unit-cell parameters a = 127.1, b = 603.5, c = 144.7 A, and form II, with space group P2(1) and unit-cell parameters a = 260.5, b = 148.2, c = 308.0 A, beta = 102.2 degrees. Data have been recorded to 4.3 and 4.0 A resolution, respectively.
W Minor - One of the best experts on this subject based on the ideXlab platform.
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pyrimidine biosynthesis in pathogens structures and analysis of Dihydroorotases from yersinia pestis and vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: Luybov Shuvalova, J Lipowska, Charles D Miks, Krzysztof Lewinski, D R Cooper, K. Kwon, I G Shabalin, H. Zheng, W MinorAbstract:Abstract The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, Dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl- l -aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis ( Yp DHO), the plague-causing pathogen, and Vibrio cholerae ( Vc DHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum .
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Pyrimidine biosynthesis in pathogens – Structures and analysis of Dihydroorotases from Yersinia pestis and Vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Luybov Shuvalova, Charles D Miks, D R Cooper, Krzysztof Lewiński, K. Kwon, I G Shabalin, H. Zheng, W MinorAbstract:The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, Dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.
J Lipowska - One of the best experts on this subject based on the ideXlab platform.
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pyrimidine biosynthesis in pathogens structures and analysis of Dihydroorotases from yersinia pestis and vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: Luybov Shuvalova, J Lipowska, Charles D Miks, Krzysztof Lewinski, D R Cooper, K. Kwon, I G Shabalin, H. Zheng, W MinorAbstract:Abstract The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, Dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl- l -aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis ( Yp DHO), the plague-causing pathogen, and Vibrio cholerae ( Vc DHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum .
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Pyrimidine biosynthesis in pathogens – Structures and analysis of Dihydroorotases from Yersinia pestis and Vibrio cholerae
International Journal of Biological Macromolecules, 2019Co-Authors: J Lipowska, Luybov Shuvalova, Charles D Miks, D R Cooper, Krzysztof Lewiński, K. Kwon, I G Shabalin, H. Zheng, W MinorAbstract:The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, Dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.
Shruti Bhargava - One of the best experts on this subject based on the ideXlab platform.
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STEM-22. TARGETING PYRIMIDINE SYNTHESIS ACCENTUATES MOLECULAR THERAPY RESPONSE IN GLIOBLASTOMA STEM CELLS
Neuro-Oncology, 2019Co-Authors: Kailin Yang, Xiuxing Wang, Leo J.y. Kim, Andrew R. Morton, Ryan C. Gimple, Briana C. Prager, Yu Shi, Wenchao Zhou, Shruti BhargavaAbstract:Abstract Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamyolase, Dihydroorotase (CAD) or the critical downstream enzyme, dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity. Higher expression of pyrimidine synthesis genes portend poor prognosis of glioblastoma patients. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.
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Targeting pyrimidine synthesis accentuates molecular therapy response in glioblastoma stem cells.
Science translational medicine, 2019Co-Authors: Xiuxing Wang, Kailin Yang, Leo J.y. Kim, Andrew R. Morton, Ryan C. Gimple, Briana C. Prager, Yu Shi, Wenchao Zhou, Shruti BhargavaAbstract:Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, Dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.
David R. Evans - One of the best experts on this subject based on the ideXlab platform.
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The crystal structure of a novel, latent Dihydroorotase from Aquifex aeolicus at 1.7A resolution.
Journal of molecular biology, 2005Co-Authors: Philip D. Martin, Cristina Purcarea, Pengfei Zhang, Asmita Vaishnav, Sharon Sadecki, Hedeel Guy-evans, David R. Evans, Brian F.p. EdwardsAbstract:Dihydroorotases (EC 3.5.2.3) catalyze the reversible cyclization of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The X-ray structures of Aquifex aeolicus Dihydroorotase in two space groups, C 222 1 and C 2, were determined at a resolution of 1.7 A. These are the first structures of a type I Dihydroorotase, a class of molecules that includes the Dihydroorotase domain of mammalian CAD. The type I enzymes are more ancient and larger, at 45 kDa, than the type II enzymes exemplified by the 38 kDa Escherichia coli Dihydroorotase. Both Dihydroorotases are members of the metallo-dependent hydrolase superfamily, whose members have a distorted “TIM barrel” domain containing the active site. However, A. aeolicus Dihydroorotase has a second, composite domain, which the E. coli enzyme lacks and has only one of the two zinc atoms present in the E. coli enzyme. A. aeolicus Dihydroorotase is unique in exhibiting significant activity only when complexed with aspartate transcarbamoylase, whereas the E. coli Dihydroorotase and the CAD Dihydroorotase domain are active as free proteins. The latency of A. aeolicus Dihydroorotase can be related to two differences between its structure and that of E. coli Dihydroorotase: (1) the monoclinic structure has a novel cysteine ligand to the zinc that blocks the active site and possibly functions as a “cysteine switch”; and (2) active site residues that bind the substrate in E. coli Dihydroorotase are located in disordered loops in both crystal structures of A. aeolicus Dihydroorotase and may function as a disorder-to-order “entropy switch”.
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Aquifex aeolicus Dihydroorotase ASSOCIATION WITH ASPARTATE TRANSCARBAMOYLASE SWITCHES ON CATALYTIC ACTIVITY
The Journal of biological chemistry, 2004Co-Authors: Anupama Ahuja, Cristina Purcarea, Sharon Sadecki, Richard Ebert, Hedeel I. Guy, David R. EvansAbstract:Abstract Dihydroorotase (DHOase) catalyzes the reversible condensation of carbamoyl aspartate to form dihydroorotate in de novo pyrimidine biosynthesis. The enzyme from Aquifex aeolicus, a hyperthermophilic organism of ancient lineage, was cloned and expressed in Escherichia coli. The purified protein was found to be a 45-kDa monomer containing a single zinc ion. Although there is no other DHOase gene in the A. aeolicus genome, the recombinant protein completely lacked catalytic activity at any temperature tested. However, DHOase formed an active complex with aspartate transcarbamoylase (ATCase) from the same organism. Whereas the kcat of 13.8 ± 0.03 s–1 was close to the value observed for the mammalian enzyme, the K mfor dihydroorotate, 3.03 ± 0.05 mm was 433-fold higher. Gel filtration and chemical cross-linking showed that the complex exists as a 240-kDa hexamer (DHO3-ATC3) and a 480-kDa duodecamer (DHO6-ATC6) probably in rapid equilibrium. Complex formation protects both DHOase and ATCase against thermal degradation at temperatures near 100 °C where the organism grows optimally. These results lead to the reclassification of both enzymes: ATCase, previously considered a Class C homotrimer, now falls into Class A, whereas the DHOase is a Class 1B enzyme. CD spectroscopy indicated that association with ATCase does not involve a significant perturbation of the DHOase secondary structure, but the visible absorption spectrum of a Co2+-substituted DHOase is appreciably altered upon complex formation suggesting a change in the electronic environment of the active site. The association of DHOase with ATCase probably serves as a molecular switch that ensures that free, uncomplexed DHOase in the cell remains inactive. At pH 7.4, the equilibrium ratio of carbamoyl aspartate to dihydroorotate is 17 and complex formation may drive the reaction in the biosynthetic direction.
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Cloning, expression and preliminary X-ray analysis of the Dihydroorotase from the hyperthermophilic eubacterium Aquifex aeolicus
Acta Crystallographica Section D Biological Crystallography, 2001Co-Authors: Cristina Purcarea, Brian F.p. Edwards, Hedeel I. Guy, P. D. Martin, John F. Vickrey, David R. EvansAbstract:Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway. The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced. The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni2+ affinity chromatography. Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique. Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li2SO4, 10 mM NiCl2, 100 mM Tris acetate pH 8.5 as the precipitant. Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension. The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, β = 99.0° and a mosaic spread of 0.6°. There is one DHOase monomer in the asymmetric unit.
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Cloning, expression and preliminary X-ray analysis of the Dihydroorotase from the hyperthermophilic eubacterium Aquifex aeolicus.
Acta crystallographica. Section D Biological crystallography, 2001Co-Authors: Cristina Purcarea, Brian F.p. Edwards, Hedeel I. Guy, John F. Vickrey, Phillip Martin, David R. EvansAbstract:Dihydroorotase (DHOase) catalyzes the formation of dihydroorotate in the de novo pyrimidine biosynthetic pathway. The gene encoding the type I DHOase from the hyperthermophilic bacterium Aquifex aeolicus has been cloned in Escherichia coli with a polyhistidine affinity tag appended to the amino-terminal end and sequenced. The recombinant protein was expressed at high levels and could be purified readily in a single step by Ni(2+) affinity chromatography. Both native and selenomethionine-labeled proteins were crystallized using the hanging-drop vapor-diffusion technique. Screens of the purified protein identified several conditions that yielded crystals; however, the best crystals were obtained using 1 M Li(2)SO(4), 10 mM NiCl(2), 100 mM Tris acetate pH 8.5 as the precipitant. Well formed diamond-shaped crystals appeared within 1 d and continued to grow over several weeks to about 0.5 mm in the largest dimension. The crystals diffract to 1.7 A and belong to space group C2, with unit-cell parameters a = 119.8, b = 88.0, c = 55.2 A, beta = 99.0 degrees and a mosaic spread of 0.6 degrees. There is one DHOase monomer in the asymmetric unit.
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Function of Conserved Histidine Residues in Mammalian Dihydroorotase
Biochemistry, 1995Co-Authors: Barbara H. Zimmermann, Nancy M. Kemling, David R. EvansAbstract:Dihydroorotase (DHOase, EC 3.5.2.3) catalyzes the reversible cyclization of carbamyl aspartate to form dihydroorotate, the third step in de novo pyrimidine biosynthesis. In mammals this activity is carried by the zinc-containing domain of the 243 kDa multifunctional protein CAD. We have replaced conserved residues in the cloned 46 kDa DHOase domain by site-directed mutagenesis. Mutants His1471Ala and His1473Ala lacked catalytic activity, judging by their failure to complement a DHOase-deficient Escherichia coli strain, and were unable to coordinate the active site zinc ion in zinc blotting experiments. This result confirmed earlier predictions. A mutant protein in which the third suspected zinc ligand was changed, Glu1512Asn, had a kcat similar to that of the intact CAD molecule and a Km similar to that of the wild-type recombinant DHOase, observations that argue against a role for glutamate 1512 in catalysis. Mutant His1590Asn had no measurable catalytic activity. This histidine residue was tentatively identified as the third zinc ligand by the failure of the mutant to bind the full complement of zinc in atomic absorption measurements. Mutant His1690Asn had a kcat 34-fold lower and a Km 9-fold higher than those of wild-type recombinant. The kinetic parameters of the mutant His1642Asn were also altered, but to a lesser extent. Diethyl pyrocarbonate (DEPC) was shown previously to inactivate mammalian DHOase. Spectroscopic studies and [14C]DEPC incorporation demonstrated that the loss of activity is associated with the modification of approximately two histidine residues located at or near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)