Thymidylate synthase (TS, EC 2.1.1.45) catalyzes the reductive methylation of dUMP by CH2H4folate to produce dTMP and H2folate. Knowledge of the catalytic mechanism and structure of TS has increased substantially over recent years. Major advances were derived from crystal structures of TS bound to various ligands, the ability to overexpress TS in heterologous hosts, and the numerous mutants that have been prepared and analyzed. These advances, coupled with previous knowledge, have culminated in an in-depth understanding of many important molecular details of the reaction. We review aspects of TS catalysis that are most pertinent to understanding the current status of the structure and catalytic mechanism of the enzyme. Included is a discussion of available sources and assays for TS, a description of the enzyme's chemical mechanism and crystal structure, and a summary of data obtained from mutagenesis experiments.

A study of the properties of the complex containing 5-fluoro-2 '-deoxyuridylate (FdUMP), 5,10-methylenetetrahydrofolate, and thymidylate synthetase is described. In the presence of the cofactor, isolable complexes contain two tightly bound molecules of FdUMP per enzyme molecule of 70,000 daltons. A number of folate analogs also stimulate binding of FdUMP, albeit to a lesser degree than the cofactor. Kinetic data indicate the rate constant for association of FdUMP with the enzyme-methylenetetrahydrofolate complex to be 2 X 107 m_1 min""* 1 at 24°. The unimolecular dissociation rates of FdUMP from the complex are highly temperature dependent and show * = 21.5 kcal/mol, /7= = 28.4 kcal/mol, and 5* = 0.023 eu; there is no indication that homotropic interactions, if existent, are manifested in the rate of dissociation of FdUMP. From kinetic data, an assocation constant for the interaction of FdUMP with the enzyme-cofactor complex is calculated to be ca. 2 X 1010 nt1 at 24°. Within the enzyme-cofactor-FdUMP complex, a t From the

DNA containing 5-azacytosine (azaC) has previously been shown to be a potent inhibitor of DNA-cytosine methyltransferases. In this report, we describe experiments which demonstrate that azaC-DNA forms a covalent complex with Hpa II methylase, a bacterial enzyme that methylates the internal C of C-C-G-G sequences. The complex does not undergo detectable dissociation over at least 3 days and is stable to denaturation with NaDodSO4. After extensive digestion of the complex with DNase and phosphodiesterase, gel filtration gave the methylase bound to approximately one equivalent of azaC; the digested complex had an apparent molecular weight similar to that of the native enzyme. Although prior treatment of azaC-DNA with Hpa II endonuclease had only a slight effect on binding of the methylase, treatment with Msp I endonuclease, which also cleaves at C-C-G-G sequences, resulted in a significant reduction in binding; this indicates that azaC residues in the recognition sequence of Hpa II are an important component in the covalent interaction of the methylase. However, since there was residual binding it is possible that azaC residues elsewhere in DNA also covalently bind to the methylase. These results provide an explanation of why azaC-DNA is such a potent inhibitor of cytosine methyltransferases and how the incorporation of such low levels of azaC into DNA can result in dramatic decreases in the methylation of cytosine. Finally, consideration of the probable catalytic mechanism of cytosine methylases and the chemical properties of azaC suggests that the inhibition is, at least in part, an active-site directed process and permits a proposal for the structure of the covalent complex.5-Methylcytosine, a minor base in the DNA of a variety of organisms, is formed by postreplicative methylation of DNA by S-adenosylmethionine (AdoMet) in reactions catalyzed by DNA-cytosine methyltransferases (DCMTases). In recent years, much evidence has been obtained which indicates that 5-methylcytosine residues in DNA play an important role in eukaryotic gene expression (for reviews see refs. 1 and 2). Consequently, there has been wide interest in the pyrimidine analog 5-azacytidine (azaCyd), which inhibits formation of 5-methylcytosine in DNA and results in dramatic effects on gene expression and cell differentiation (e.g., see refs. 3-9).Current evidence indicates that the mechanism by which azaCyd causes decreased DNA methylation involves incorporation of 5-azacytosine (azaC) into DNA and subsequent inhibition of DCMTase. Incorporation of small amounts of azaC into DNA of mammalian cells results in a loss of DCMTase activity in extracts obtained from such cells (5,10). Further, incubation of DNA containing azaC (azaC-DNA) with mammalian or bacterial DCMTases results in a very potent inhibition of enzyme activity (11, 12), but kinetic studies have not revealed the mechanism of inhibition. On the basis of the probable catalytic mechanism of DCMTases and known chemical properties of azaC, we recently speculated that the mechanism o...

Single and multiple mutations at residues 16, 51, 59, 108, and 164 of Plasmodium falciparum dihydrofolate reductase (pfDHFR) have been linked to antifolate resistance in malaria. We prepared and characterized all seven of the pfDHFR mutants found in nature, as well as six mutants not observed in nature. Mutations involving residues 51, 59, 108, or 164 conferred cross resistance to both the antifolates pyrimethamine and cycloguanil, whereas mutation of residue 16 specifically conferred resistance to cycloguanil. The antifolate resistance of enzyme mutants found in nature correlated with in vivo antifolate resistance; however, mutants not found in nature were either poorly resistant or had insufficient catalytic activity to support DNA synthesis. Thus, specific combinations of multiple mutations at target residues were selected in nature to optimize resistance. Further, the resistance of multiple mutants was more than the sum of the component single mutations, indicating that residues were selected for their synergistic as well as intrinsic effects on resistance. Pathways inferred for the evolution of pyrimethamine-resistant mutants suggested that all multiple mutants emerged from stepwise selection of the single mutant, S108N. Thus, we propose that drugs targeted to both the wild-type pfDHFR and S108N mutant would have a low propensity for developing resistance, and hence could provide effective antimalarial agents.

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.