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Query: UNIPROT:P06889 (Mol)
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Thymidylate synthase (TS) is a homodimeric enzyme that catalyzes the reductive methylation of dUMP by N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, to form dTMP. Inhibition of TS by the dUMP analog 5-fluoro-dUMP (FdUMP) occurs through the formation of a covalent ternary complex containing the nucleotide analog, N5,N10-methylene-5,6,7,8-tetrahydrofolic acid, and the enzyme; this complex is termed the inhibitory ternary complex (ITC). In the present report, the kinetics of FdUMP binding into an ITC with purified preparations of human TS were examined. Rapid chemical-quench techniques, as well as steady state binding methods, showed that the enzyme contains two distinct FdUMP binding sites with different affinities for the nucleotide analog. Binding to the first, or high affinity, site was rapid and reached a maximum stoichiometry of 1.0 mol of FdUMP/mol of dimer; binding to the second, or low affinity, site was much slower and reached a stoichiometry of 1.7 mol of FdUMP/mol of dimer. Rate constants for FdUMP binding to and dissociation from the ITC (kon and koff, respectively) were determined, as were equilibrium dissociation constants (Kd). A naturally occurring mutant form of TS, which contains a tyrosine to histidine substitution at residue 33 and renders cells relatively resistant to fluoropyrimidines, exhibited a lower affinity for FdUMP specifically at the second binding site, with little or no change at the first. Hill coefficients were < 1.0, with the His-33 enzyme having a significantly lower coefficient than the wild-type enzyme. The results, in total, indicate that the two FdUMP binding sites on the TS dimer are nonequivalent. We suggest that such nonequivalence may be due to negative cooperativity, where nucleotide binding to the first subunit elicits a conformational change that results in reduced affinity for ligand at the second subunit. This negative cooperativity may be stronger for the His-33 mutant. Thus, the relative fluoropyrimidine resistance conferred by the His-33 substitution may be due to enhanced negative cooperative effects on FdUMP binding into the ITC, thereby reducing the effectiveness of the pyrimidine analog as an inhibitor of thymidylate biosynthesis.
Mol Pharmacol 1995 Jul
PMID:Biphasic binding of 5-fluoro-2'-deoxyuridylate to human thymidylate synthase. 762 77

We have found that Bacillus subtilis possesses a second 5'-phosphoribosyl-1-glycinamide (GAR) transformylase catalysing the first one-carbon transfer reaction in the purine biosynthetic pathway. Inactivation of the purN gene encoding the N10-formyl tetrahydrofolate-dependent enzyme did not result in purine auxotrophy. However, growth of a purN strain was stimulated when either purine or formate was added to the growth medium. In cell-free extracts GAR could be formylated, provided formate was added to the assay mixture. From the purN strain, purine-requiring mutants were isolated. One of these mutant strains was defective in the formate-dependent formylation of GAR in vitro. The gene containing this second mutation was designated purT, and was mapped to approximately 20 degrees on the genetic map between the cysA and aroI markers.
Mol Gen Genet 1994 Feb
PMID:Genetic and physiological characterization of a formate-dependent 5'-phosphoribosyl-1-glycinamide transformylase activity in Bacillus subtilis. 812 96

The in vitro metabolism of cortisol in human liver fractions is highly complex and variable. Cytosolic metabolism proceeds predominantly via A-ring reduction (to give 3 alpha,5 beta-tetrahydrocortisol; 3 alpha,5 beta-THF), while microsomal incubations generate upto 7 metabolites, including 6 beta-hydroxycortisol (6 beta-OHF), and 6 beta-hydroxycortisone (6 beta-OHE), products of the cytochrome P450 (CYP) 3A subfamily. The aim of the present study was, therefore, to examine two of the main enzymes involved in cortisol metabolism, namely, microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase. In particular, we wished to assess the substrate specificity of these enzymes and identify compounds with inhibitory potential. Incubations for 30 min containing [3H]cortisol, potential inhibitors, microsomal or cytosolic protein (3 mg), and co-factors were followed by radiometric HPLC analysis. The Km value for 6 beta-OHF and 6 beta-OHE formation was 15.2 +/- 2.1 microM (mean +/- SD; n = 4) and the Vmax value 6.43 +/- 0.45 pmol/min/mg microsomal protein. The most potent inhibitor of cortisol 6 beta-hydroxylase was ketoconazole (Ki = 0.9 +/- 0.4 microM; n = 4), followed by gestodene (Ki = 5.6 +/- 0.6 microM) and cyclosporine (Ki = 6.8 +/- 1.4 microM). Both betamethasone and dexamethasone produced some inhibition (Ki = 31.3 and 54.5 microM, respectively). However, substrates for CYP2C (tolbutamide), CYP2D (quinidine), and CYP1A (theophylline) were essentially non-inhibitory. The Km value for cortisol 4-ene-reductase was 26.5 +/- 11.2 microM (n = 4) and the Vmax value 107.7 +/- 46.0 pmol/min/mg cytosolic protein. The most potent inhibitors were androstendione (Ki = 17.8 +/- 3.3 microM) and gestodene (Ki = 23.8 +/- 3.8 microM). Although both compounds have identical A-rings to cortisol, and undergo reduction, inhibition was non-competitive.
J Steroid Biochem Mol Biol 1993 Dec
PMID:Cortisol metabolism in vitro--III. Inhibition of microsomal 6 beta-hydroxylase and cytosolic 4-ene-reductase. 827 18

The genomes of several thermophilic members of the genus Methanobacterium were analyzed for homology to the related restriction-modification plasmids pFV1 and pFZ1 from M. thermoformicicum strains THF and Z-245, respectively. Two plasmid regions, designated FR-I and FR-II, could be identified with chromosomal counterparts in six Methanobacterium strains. Multiple copies of the pFV1-specific element FR-I were detected in the M. thermoformicicum strains CSM3, FF1, FF3 and M. thermoautotrophicum delta H. Sequence analysis showed that one FR-I element had been integrated in almost identical sequence contexts into the chromosomes of the strains CSM3 and delta H. Comparison of the FR-I elements from these strains with that from pFV1 revealed that they consisted of two subfragments, boxI (1118 bp) and boxII (383 bp), the order of which is variable. Each subfragment was identical on the sequence level with the corresponding plasmid-borne element and was flanked by terminal direct repeats with the consensus sequence A(A/T)ATTT. These results suggest that FR-I represents a mobile element. FR-II was located on both plasmids pFV1 and pFZ1, and on the chromosome of M. thermoformicicum strains THF, CSM3 and HN4. Comparison of the nucleotide sequences of the two plasmid FR-II copies and that from the chromosome of strain CSM3 showed that the FR-II segments were approximately 2.5-3.0 kb in size and contained large open reading frames (ORFs) that may encode highly related proteins with an as yet unknown function.
Mol Gen Genet 1993 Jul
PMID:Distribution and characterization of plasmid-related sequences in the chromosomal DNA of different thermophilic Methanobacterium strains. 839 14

The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cytoplasmic trifunctional C1-tetrahydrofolate (THF) synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) activities. However, it has been suggested that the ADE3-encoded C1-THF synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosynthesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis [Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687]. This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF synthase is enough to complement the adenine requirement in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the C1-THF synthase is involved in purine biosynthesis.
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PMID:Function of yeast cytoplasmic C1-tetrahydrofolate synthase. 846 69

Thymine-requiring strains of Escherichia coli suppress nonsense and frameshift mutants of T4 phage. We proposed that these mutants make errors during translation because of an imbalance in folate metabolism. A thymine-requiring strain grown under suppressing conditions has elevated levels of reduced folates. We tested the effect of either mutational blocks or the inhibition of various steps in folate biosynthesis on suppression. Conditions which prevent the accumulation of 5-methyl tetrahydrofolate inhibit suppression, suggesting that elevated levels of this folate are required for suppression. Furthermore, conditions that result in an accumulation in dihydrofolate inhibit suppression.
Mol Gen Genet 1993 Apr
PMID:Nonsense suppression in thymine-requiring strains of Escherichia coli is a consequence of altered folate metabolism. 847 27

Two human colorectal tumor cell lines are differentially sensitive to growth inhibition by 5-fluorodeoxyuridine (FdUrd); cell line RCA is less sensitive to FdUrd than is cell line C. Thymidylate synthase (TS), a target of FdUrd, has been purified to homogeneity from both cell lines. Because of differences in the avidity for a folate ligand affinity matrix, TS forms from the cells were purified by two different procedures. Relative to the enzyme from C cells, the enzyme from RCA cells demonstrated higher Km values for the substrates deoxyuridylate and 5,10-methylene-tetrahydrofolate, a lower rate of association of the inhibitor 5-fluorodeoxyuridylate (FdUMP), a similar rate of FdUMP dissociation, and lower enhancement of covalent FdUMP binding by folate derivatives. The activities of the enzymes in situ and the catalytic efficiencies of the purified enzymes were similar. Thus, a cell line that is naturally resistant to FdUrd has been identified that expresses a TS with reduced affinity for FdUMP and 5,10-methylenetetrahydrofolate, relative to the enzyme expressed in a FdUrd-sensitive cell line.
Mol Pharmacol 1993 May
PMID:Variation in human thymidylate synthase is associated with resistance to 5-fluoro-2'-deoxyuridine. 850 27

Three steps along the pathway of binding, orientation of substrates and release of products are revealed by X-ray crystallographic structures of ternary complexes of the wild-type Lactobacillus casei thymidylate synthase enzyme. Each complex was formed by diffusion of either the cofactor 5,10-methylene-5,6,7,8-tetrahydrofolate or the folate analog 10-propargyl-5,8-dideazafolate into binary co-crystals of thymidylate synthase with 2'-deoxyuridine-5'-monophosphate. A two-substrate/enzyme complex is formed where the substrates remain unaltered. The imidazolidine ring is unopened and the pterin of the 5,10-methylene-5,6,7,8-tetrahydrofolate cofactor binds at an unproductive "alternate" site. We propose that the presence of the pterin at this site may represent an initial interaction with the enzyme that precedes all catalytic events. The structure of the 2'-deoxyuridine-5'-monophosphate and 10-propargyl-5,8-dideazafolate folate analog complex identifies both ligands in orientations favorable for the initiation of catalysis and resembles the productive complex. A product complex where the ligands have been converted into products of the thymidylate synthase reaction within the crystal, 2'-deoxythymidine-5'-monophosphate and 7,8-dihydrofolate, shows how ligands are situated within the enzyme after catalysis and on the way to product release.
J Mol Biol 1996 Jan 26
PMID:Entropy in bi-substrate enzymes: proposed role of an alternate site in chaperoning substrate into, and products out of, thymidylate synthase. 856 95

Periconceptional folate intake reduces both the occurrence and recurrence risk of neural tube defects. Plasma homocysteine levels can be elevated in mothers of a child with a neural tube defect, suggesting a dysfunctional folate metabolism. Very recently we showed that a common 677C-->T mutation in the 5,10-methylene tetrahydrofolate reductase gene, causing thermolability of the enzyme, is a risk factor for spina bifida offspring. Restriction enzyme analysis of the genomic 5,10-methylene tetrahydrofolate reductase polymerase chain reaction fragment revealed a significantly higher prevalence of a +/+ genotype among spina bifida patients and their mothers. The risk for spina bifida offspring is the strongest if both the mother and her child have the mutation in the homozygous state. Enzymatic analysis showed that homozygosity for the 677C-->T mutation causes a decreased 5,10-methylene tetrahydrofolate reductase activity, resulting in elevated plasma homocysteine and red blood cell folate levels and lowered plasma folate and cysteine values. This extended study demonstrates that a nucleotide substitution in the coding region of 5,10-methylene tetrahydrofolate reductase, resulting in reduced activity and an impaired homocysteine and folate metabolism, is a genetic risk factor for spina bifida.
J Mol Med (Berl) 1996 Nov
PMID:Decreased methylene tetrahydrofolate reductase activity due to the 677C-->T mutation in families with spina bifida offspring. 895 55

We have determined structures of binary and ternary complexes of five Asn229 variants of thymidylate synthase (TS) and related their structures to the kinetic constants measured previously. Asn229 forms two hydrogen bonds to the pyrimidine ring of the substrate 2'-deoxyuridine-5'-monophosphate (dUMP). These hydrogen bonds constrain the orientation of dUMP in binary complexes with dUMP, and in ternary complexes with dUMP and the TS cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. In N229 mutants, where these hydrogen bonds cannot be made, dUMP binds in a misoriented or more disordered fashion. Most N229 mutants exhibit no activity for the dehalogenation of 5-bromo-dUMP, which requires correct orientation of dUMP against Cys198. Since bound dUMP forms the binding surface against which the pterin ring of cofactor binds, misorientation of dUMP results in higher Km values for cofactor. At the same time, binding of the cofactor aids in ordering and positioning dUMP for catalysis. Hydrophobic mutants, such as N229I, favor an arrangement of solvent molecules and side-chains around the ligands similar to that in a proposed transition state for ternary complex formation in wild-type TS, and kcat values are similar to the wild-type value. Smaller, more hydrophilic mutants favor arrangements of the solvent and side-chains surrounding the ligands that do not resemble the proposed transition state. These changes correspond to decreases in kcat of up to 2000-fold, with only modest increases in Km or Kd. These results are consistent with the proposal that the hydrogen-bonding network between water, dUMP and side-chains in the active-site cavity contributes to catalysis in TS. Asn229 has the unique ability to maintain this critical network, without sterically interfering with dUMP binding.
J Mol Biol 1998 Feb 13
PMID:Contributions of orientation and hydrogen bonding to catalysis in Asn229 mutants of thymidylate synthase. 951 16


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