No abstract

Synthesis of dTMP by thymidylate synthase proceeds by the reductive methylation of dUMP, which is obtained via one of two parallel pathways. One pathway, considered to be a minor supplier of dTTP [1][2][3], involves the reduction of UDP (UTP) by the action of ribonucleotide reductase. Subsequently, dUDP is phosphorylated to dUTP and cleaved to dUMP. The main supply of dUMP, however, involves the deamination The trimeric dCTP deaminase produces dUTP that is hydrolysed to dUMP by the structurally closely related dUTPase. This pathway provides 70-80% of the total dUMP as a precursor for dTTP. Accordingly, dCTP deaminase is regulated by dTTP, which increases the substrate concentration for half-maximal activity and the cooperativity of dCTP saturation. Likewise, increasing concentrations of dCTP increase the cooperativity of dTTP inhibition. Previous structural studies showed that the complexes of inactive mutant protein, E138A, with dUTP or dCTP bound, and wild-type enzyme with dUTP bound were all highly similar and characterized by having an ordered C-terminal. When comparing with a new structure in which dTTP is bound to the active site of E138A, the region between Val120 and His125 was found to be in a new conformation. This and the previous conformation were mutually exclusive within the trimer. Also, the dCTP complex of the inactive H121A was found to have residues 120-125 in this new conformation, indicating that it renders the enzyme inactive. The C-terminal fold was found to be disordered for both new complexes. We suggest that the cooperative kinetics are imposed by a dTTP-dependent lag of product formation observed in presteady-state kinetics. This lag may be derived from a slow equilibration between an inactive and an active conformation of dCTP deaminase represented by the dTTP complex and the dUTP ⁄ dCTP complex, respectively. The dCTP deaminase then resembles a simple concerted system subjected to effector binding, but without the use of an allosteric site.Abbreviations E138A, mutant dCTP deaminase with a Glu138 to Ala substitution; H121A, mutant dCTP deaminase with a His121 to Ala substitution; V122G, mutant dCTP deaminase with a Val122 to Gly substitution.

Dihydroorotate dehydrogenases (DHODs) catalyze the oxidation of dihydroorotate to orotate in the only redox reaction in pyrimidine biosynthesis. The pyrimidine binding sites are very similar in all structurally characterized DHODs, suggesting that the prospects for identifying a class-specific inhibitor directed against this site are poor. Nonetheless, two compounds that bind specifically to the Class 1A DHOD from Lactococcus lactis, 3,4-dihydroxybenzoate (3,4-diOHB) and 3,5-dihydroxybenzoate (3,5-diOHB), have been identified [Palfey et al. (2001) J. Med. Chem. 44, 2861-2864]. The mechanism of inhibitor binding to the Class 1A DHOD from L. lactis has now been studied in detail and is reported here. Titrations showed that 3,4-diOHB binds more tightly at higher pH, whereas the opposite is true for 3,5-diOHB. Isothermal titration calorimetry and absorbance spectroscopy showed that 3,4-diOHB ionizes to the phenolate upon binding to the enzyme, but 3,5-diOHB does not. The charge-transfer band that forms in the 3,4-diOHB complex allowed the kinetics of binding to be observed in stopped-flow experiments. Binding was slow enough to observe from pH 6 to pH 8 and was (minimally) a two-step process consisting of the rapid formation of a complex that isomerized to the final charge-transfer complex. Orotate and 3,5-diOHB bind too quickly to follow directly, but their dissociation kinetics were studied by competition and described adequately with a single step. Crystal structures of both inhibitor complexes were determined, showing that 3,5-diOHB binds in the same orientation as orotate. In contrast, 3,4-diOHB binds in a twisted orientation, enabling one of its phenolic oxygens to form a very strong hydrogen bond to an asparagine, thus stabilizing the phenolate and causing charge-transfer interactions with the pi-system of the flavin, resulting in a green color.