The flavoenzymes dihydroorotate dehydrogenases (DHODs) catalyze the fourth and only redox step in the de novo biosynthesis of UMP. Enzymes belonging to class 2, according to their amino acid sequence, are characterized by having a serine residue as the catalytic base and a longer N terminus. The structure of class 2 E. coli DHOD, determined by MAD phasing, showed that the N-terminal extension forms a separate domain. The catalytic serine residue has an environment differing from the equivalent cysteine in class 1 DHODs. Significant differences between the two classes of DHODs were identified by comparison of the E. coli DHOD with the other known DHOD structures, and differences with the class 2 human DHOD explain the variation in their inhibitors.

The flavin-containing enzyme dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate (DHO) to orotate, the first aromatic intermediate in pyrimidine biosynthesis. The first structure of a DHOD, the A form of the enzyme from Lactococcus lactis, has recently become known, and some conserved residues were suggested to have a role in the active site [Rowland et al. (1997) Structure 2, 239-252]. In particular, Cys 130 was hypothesized to work as a base, which activates dihydroorotate (DHO) for hydride transfer. By chemical modification and site-directed mutagenesis we have obtained results consistent with this proposal. Cys 130 was susceptible to alkylating reagents, and mutants of Cys 130 (C130A and C130S) showed hardly detectable enzyme activity at pH 8.0, while at pH 10 the C130S mutant enzyme had approximately 1% of wild-type activity. Mutants of Lys 43, Asn 132, and Lys 164 were also constructed. Exchange of Lys 43 to Ala or Glu (K43A and K43E) and of Asn 132 to Ala (N132A) affected both catalysis and substrate binding. Expressed as kcat/KM for DHO, the deterioration of these three mutant enzymes was 10(3)-10(4)-fold. Flavin spectra of the mutant enzymes were not, like the wild-type enzyme, bleached by DHO in stopped-flow experiments, showing that they were deficient with respect to the first half-reaction, namely reduction of FMN by DHO, which was not rate limiting for the wild-type enzyme. The binding interaction between flavin and the reaction product, orotate, could be monitored by a red shift of the flavin absorbance in the wild-type enzyme. The C130A, C130S, and N132A mutant enzymes displayed similar capacity to bind orotate. In contrast, orotate did not change the absorption spectra of the K43 mutant enzymes, although it did inhibit their activity. All of the mutant enzymes, except K164A, contained normal levels of flavin. The results are discussed in relation to the structures of DHODA and other flavoenzymes. The possible acid-base chemistry of Cys 130 is compared to previous work on mammalian dihydropyrimidine dehydrogenases, flavoenzymes, which catalyze the reversed reaction, namely the reduction of pyrimidine bases.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of (S)-dihydroorotate to orotate, the fourth step and only redox reaction in the de novo biosynthesis of pyrimidine nucleotides. A description is given of the crystal structure of Lacrococcus lacris dihydroorotate dehydrogenase A (DHODA) complexed with the product of the enzyme reaction orotate. The structure of the complex to 2.0 8, resolution has been compared with the structure of the native enzyme.The active site of DHODA is known to contain a water filled cavity buried beneath a highly conserved and flexible loop. In the complex the orotate displaces the water molecules from the active site and stacks above the DHODA flavin isoalloxazine ring, causing only small movements of the surrounding protein residues. The orotate is completely buried beneath the protein surface, and the orotate binding causes a significant reduction in the mobility of the active site loop. The orotate is bound by four conserved asparagine side chains (Asn 67, Asn 127, Asn 132, and Asn 193), the side chains of Lys 43 and Ser 194, and the main chain NH groups of Met 69, Gly 70, and Leu 71. Of these the Lys 43 side chain makes hydrogen bonds to both the flavin isoalloxazine ring and the carboxylate group of the orotate. Potential interactions with bound dihydroorotate are considered using the orotate complex as a basis for molecular modeling. The role of Cys 130 as the active site base is discussed, and the sequence conservation of the active site residues across the different families of DHODs is reviewed, along with implications for differences in substrate binding and in the catalytic mechanisms between these families.

Dihydroorotate dehydrogenase (DHOD) oxidizes dihydroorotate (DHO) to orotate in the only redox reaction of pyrimidine biosynthesis. The enzyme from Escherichia coli is a membrane-bound FMN-containing enzyme that is thought to use ubiquinone as the oxidizing substrate. The chemistry of the reduction of the flavin in DHOD from E. coli by the substrate dihydroorotate (DHO) was studied at 4 degrees C in anaerobic stopped-flow experiments conducted over a broad range of pH values. A Michaelis complex that was characterized by a approximately 20 nm red-shift of the oxidized flavin absorbance formed within the dead-time of the stopped-flow instrument ( approximately 1 ms) upon mixing with DHO. The flavin of the intermediate was reduced by DHO, forming a reduced flavin-orotate charge-transfer complex. The rate constant for the flavin reduction reaction increased with pH, from a value of 1 s(-1) at pH 6.5 to approximately 360 s(-1) at pH values greater than an observed pK(a) of 9.5 which was ascribed to Ser175, the active-site base. At all pH values, the reduced flavin-orotate charge-transfer complex dissociated too slowly to be catalytically relevant. Therefore, the oxidizing quinone substrate must bind to the reduced enzyme-orotate complex at a site distinct from the substrate binding site, in agreement with steady-state kinetic studies [Björnberg, O., Grüner, A.-C., Roepstorff, P., and Jensen, K. F. (1999) Biochemistry 38, 2899-2908]. Menadione was used as a model quinone substrate to oxidize dithionite-reduced DHOD. The reduced enzyme-orotate complex reacted rapidly with menadione (180 s(-1)), demonstrating that the reduced enzyme-orotate complex is a catalytically competent intermediate.