Functional identity of the active sites of crustacean and viral thymidylate synthases.

Thymidylate synthase (TS) catalyzes the synthesis of deoxythymidine monophosphate (dTMP), which is an essential precursor for DNA synthesis. The rationale underlying drug design is to identify compounds that differentially inhibit a viral or parasite enzyme vs. the host homologue.

Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS.

Role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase.

Thymidylate synthase (TS) catalyzes the substitution of a carbon-bound proton in a uracil base by a methyl group to yield thymine in the de novo biosynthesis of this DNA base. The enzymatic mechanism involves making and breaking several covalent bonds. Traditionally, a conserved tyrosine (Y94 in Escherichia coli, Y146 in Lactobacillus casei, and Y135 in humans) was assumed to serve as the general base catalyzing the proton abstraction.

Significance of mutations on the structural perturbation of thymidylate synthase: implications for their involvement in subunit exchange.

Wild-type thymidylate synthase (WT-TS) from Escherichia coli and several of its mutants showed varying degrees of susceptibility to trypsin. While WT-TS was resistant to trypsin as were the mutants C146S, K48E, and R126K, others such as Y94A, Y94F, C146W, and R126E were digested but at different rates from one another. The peptides released from the mutants were identified by mass spectrometry and Edman sequence analysis.

Chloroviruses encode a bifunctional dCMP-dCTP deaminase that produces two key intermediates in dTTP formation.

The chlorovirus PBCV-1, like many large double-stranded DNA-containing viruses, contains several genes that encode putative proteins involved in nucleotide biosynthesis. This report describes the characterization of the PBCV-1 dCMP deaminase, which produces dUMP, a key intermediate in the synthesis of dTTP. As predicted, the recombinant protein has dCMP deaminase activity that is activated by dCTP and inhibited by dTTP.

Structure of the Y94F mutant of Escherichia coli thymidylate synthase.

Tyr94 of Escherichia coli thymidylate synthase is thought to be involved, either directly or by activation of a water molecule, in the abstraction of a proton from C5 of the 2'-deoxyuridine 5'-monophosphate (dUMP) substrate. Mutation of Tyr94 leads to a 400-fold loss in catalytic activity. The structure of the Y94F mutant has been determined in the native state and as a ternary complex with thymidine 5'-monophosphate (dTMP) and 10-propargyl 5,8-dideazafolate (PDDF).

Hydride transfer versus hydrogen radical transfer in thymidylate synthase.

The nature of a H-transfer in the thymidylate synthase catalyzed reaction was investigated by comparison of the wild-type enzyme with the W80M mutant. The nature of the H-transfer was not affected, as indicated by intrinsic isotope effects and their temperature dependence. These findings support a single-step hydride transfer instead of a two-step radical transfer.

Crystal structures of thymidylate synthase mutant R166Q: structural basis for the nearly complete loss of catalytic activity.

Thymidylate synthase (TS) catalyzes the folate-dependent methylation of deoxyuridine monophosphate (dUMP) to form thymidine monophosphate (dTMP). We have investigated the role of invariant arginine 166, one of four arginines that contact the dUMP phosphate, using site-directed mutagenesis, X-ray crystallography, and TS from Escherichia coli. The R166Q mutant was crystallized in the presence of dUMP and a structure determined to 2.

Binding and repression of translation of the cognate mRNA by Trichinella spiralis thymidylate synthase differ from the corresponding interactions of the human enzyme.

Thymidylate synthase (TS) of Trichinella spiralis, a parasitic nematode causing trichinellosis, was found to bind its own mRNA and repress translation of the latter, similar to its human counter-part [Chu, Koeller, Casey, Drake, Chabner, Elwood, Zinn and Allegra (1991) Proc. Natl. Acad.

Three-dimensional structure of the R115E mutant of T4-bacteriophage 2'-deoxycytidylate deaminase.

2'-Deoxycytidylate deaminase (dCD) converts deoxycytidine 5'-monophosphate (dCMP) to deoxyuridine 5'-monophosphate and is a major supplier of the substrate for thymidylate synthase, an important enzyme in DNA synthesis and a major target for cancer chemotherapy. Wild-type dCD is allosterically regulated by the end products of its metabolic pathway, deoxycytidine 5'-triphosphate and deoxythymidine 5'-triphosphate, which act as an activator and an inhibitor, respectively. The first crystal structure of a dCD, in the form of the R115E mutant of the T4-bacteriophage enzyme complexed with the active site inhibitor pyrimidin-2-one deoxyribotide, has been determined at 2.

Protection of hematopoietic stem cells from pemetrexed toxicity by retroviral gene transfer with a mutant dihydrofolate reductase-mutant thymidylate synthase fusion gene.

Myelosuppression is one of the major side effects of most anticancer drugs. To confer myeloprotection, our laboratory generated drug-resistant mutants of select target human enzymes for gene transfer to the bone marrow. Mutants of two of these enzymes, dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S), were previously shown to confer resistance to methotrexate and 5-FU, respectively, and recently a fusion cDNA of both mutant enzymes (DHFR F/S-TS G52S) was shown to confer dual resistance to both antimetabolites.

A trojan horse approach for silencing thymidylate synthase.

In this paper we present a new and possibly more effective way of inhibiting thymidylate synthase (TS) in cells than through the use of substrate analogue inhibitors. An inactive double mutant of TS (DM), Arg(126)Glu/Cys(146)Trp, is shown to progressively impair the reactivation of native Escherichia coli TS when the two are denatured together in vitro. The individual single mutant proteins Arg(126)Glu and Cys(146)Trp showed little or no inhibition.

Role of cysteine amino acid residues on the RNA binding activity of human thymidylate synthase.

The role of cysteine sulfhydryl residues on the RNA binding activity of human thymidylate synthase (TS) was investigated by mutating each cysteine residue on human TS to a corresponding alanine residue. Enzymatic activities of TS:C43A and TS:C210A mutant proteins were nearly identical to wild-type TS, while TS:C180A and TS:C199A mutants expressed >80% of wild-type enzyme activity. In contrast, TS:C195A was completely inactive.

Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells.

We have previously observed that the expression of two thymidylate biosynthesis enzymes, dihydrofolate reductase and thymidylate synthase (TS), is upregulated in quiescent human fibroblasts infected with human cytomegalovirus (HCMV). Here, we have demonstrated that HCMV increases expression of the cellular deoxycytidylate deaminase (dCMP deaminase), which provides the substrate for TS by converting dCMP to dUMP. We observed an increase in dCMP deaminase protein levels, whereas deoxyuridine triphosphatase (dUTPase), another cellular enzyme that may provide dUMP by hydrolysing dUTP, was undetectable.

Modification of Escherichia coli thymidylate synthase at tyrosine-94 by 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate.

Evidence is presented that 5-imidazolylpropynyl-2'-deoxyuridine 5'-monophosphate (IP-dUMP) is a mechanism-based, irreversible inactivator of Escherichia coli thymidylate synthase (TS), which covalently modifies Tyr94 at the active site of the enzyme. The inactivation of TS was time and concentration dependent and did not require the folate cofactor. Due to the rapidity of the inactivation process, accurate kinetic parameters could be determined only in the presence of saturating concentrations (1000K(M)) of the competing substrate, dUMP.

Retroviral transduction of a mutant dihydrofolate reductase-thymidylate synthase fusion gene into murine marrow cells confers resistance to both methotrexate and 5-fluorouracil.

Gene transfer-based myeloprotection strategies against chemotherapy require the development of effective drug resistance genes or gene combinations. Our laboratory has previously generated drug-resistant mutants of dihydrofolate reductase (DHFR F/S) and thymidylate synthase (TS G52S) for myeloprotection against methotrexate (MTX) and 5-fluorouracil (5-FU), respectively. For the purpose of conferring dual myeloprotection against both MTX and 5-FU, we have generated two retroviral constructs encoding both DHFR F/S and TS G52S as a fusion protein (DHFR F/S-TS G52S) or as individual proteins from a bicistronic gene.

Mechanistic characterization of Toxoplasma gondii thymidylate synthase (TS-DHFR)-dihydrofolate reductase. Evidence for a TS intermediate and TS half-sites reactivity.

This study describes the use of rapid transient kinetic methods to characterize the bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme from Toxoplasma gondii. In addition to elucidating the detailed kinetic scheme for this enzyme, this work provides the first direct kinetic evidence for the formation of a TS intermediate and for half-sites TS reactivity in human and Escherichia coli monofunctional TS and in T. gondii and Leishmania major bifunctional TS-DHFR.