The structure and mechanism of bacterial dihydroorotase
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Dihydroorotase (DHO) is a zinc metallo-enzyme that functions in the pathway for the biosynthesis of pyrimidine nucleotides by catalyzing the reversible interconversion of carbamoyl aspartate and dihydroorotate. The X-ray crystal structure of the enzyme was obtained at a resolution of 1.7 Ã . The pH-rate profiles for the hydrolysis of dihydroorotate or thio-dihydroorotate demonstrated that a single group of DHO must be unprotonated for maximal catalytic activity. The pH-rate profiles for the condensation of carbamoyl aspartate to dihydroorotate showed that a single group from the enzyme must be protonated for maximal catalytic activity. The native zinc ions within the active site of DHO were substituted with cobalt or CADmium by reconstitution of the apo-enzyme with divalent cations. The ionizations observed in the pH-rate profiles were dependent on the specific metal ion bound to the active site. Mutation of Asp-250 resulted in the loss of catalytic activity. These results are consistent with the formation of a hydroxide bridge between the two divalent cations that functions as the nucleophile during the hydrolysis of dihydroorotate. In addition, Asp250 is postulated to shuttle the proton from the bridging hydroxide to the leaving group amide during dihydroorotate hydrolysis. The X-ray crystal structure of DHO showed that the side-chain carboxylate of dihydroorotate is electrostatically interacting with Arg20, Asn-44 and His-254. Mutation of these residues resulted in the loss of catalytic activity, indicating that these residues are critical for substrate recognition. The thioanalog of dihydroorotate, (TDO) was found to be a substrate of DHO. A comprehensive chemical mechanism for DHO was proposed based on the experimental data presented in this dissertation. Armed with this understanding of the structure-function relationship of DHO, a rational approach was used to alter the substrate specificity of the enzyme. The R20/N44/H254 mutant of DHO was obtained and found to have increased activity on dihydrouracil compared to the wild-type enzyme. The sequence of the gene PA5541 from Pseudomonas aeruginosa has a glutamine at a position where most active DHO proteins have a histidine residue. Results from the characterization of PA5541 indicate that it is a functional DHO.
Porter, Tamiko Neal (2004). The structure and mechanism of bacterial dihydroorotase. Doctoral dissertation, Texas A&M University. Texas A&M University. Available electronically from