Crystal Structures Capture Three States in the Catalytic Cycle of a Pyridoxal Phosphate (PLP) Synthase

PLP synthase (PLPS) is a remarkable single-enzyme biosynthetic pathway that produces pyridoxal 5′-phosphate (PLP) from glutamine, ribose 5-phosphate, and glyceraldehyde 3-phosphate. The intact enzyme includes 12 synthase and 12 glutaminase subunits. PLP synthesis occurs in the synthase active site by a complicated mechanism involving at least two covalent intermediates at a catalytic lysine. The first intermediate forms with ribose 5-phosphate. The glutaminase subunit is a glutamine amidotransferase that hydrolyzes glutamine and channels ammonia to the synthase active site. Ammonia attack on the first covalent intermediate forms the second intermediate. Glyceraldehyde 3-phosphate reacts with the second intermediate to form PLP. To investigate the mechanism of the synthase subunit, crystal structures were obtained for three intermediate states of the Geobacillus stearothermophilus intact PLPS or its synthase subunit. The structures capture the synthase active site at three distinct steps in its complicated catalytic cycle, provide insights into the elusive mechanism, and illustrate the coordinated motions within the synthase subunit that separate the catalytic states. In the intact PLPS with a Michaelis-like intermediate in the glutaminase active site, the first covalent intermediate of the synthase is fully sequestered within the enzyme by the ordering of a generally disordered 20-residue C-terminal tail. Following addition of ammonia, the synthase active site opens and admits the Lys-149 side chain, which participates in formation of the second intermediate and PLP. Roles are identified for conserved Asp-24 in the formation of the first intermediate and for conserved Arg-147 in the conversion of the first to the second intermediate.     

Localization of the deoxyribonucleotide biosynthetic enzymes ribonucleotide reductase and thymidylate synthase in mouse L cells

Two different approaches were used to define the intracellular localization in mouse L929 cells of two deoxyribonucleotide biosynthetic enzymes: ribonucleoside diphosphate reductase (EC1.17.4.1) and thymidylate synthase (EC2.1.1.45). The first involved treatment with saponins, which render the plasma membrane permeable to proteins without disrupting intracellular organelles. Under conditions where nuclear DNA synthesis and the activity of the nuclear enzyme NMN adenylyltransferase were unaffected, the entire cellular complements of a cytosolic enzyme, glucose-6-phosphate dehydrogenase, and of ribonucleotide reductase and thymidylate synthase were released at the same rate and with similar dependence on saponin concentration. The second approach involved centrifugal enucleation of cells treated with cytochalasin B (CB) and measurement of the distribution of enzyme activities in the resulting cytoplast and karyoplast fractions. Whereas most NMN adenylyltransferase activity remained with the karyoplasts, glucose-6-phosphate dehydrogenase, ribonucleotide reductase, and thymidylate synthase were almost exclusively associated with the enucleated cytoplasts. These results indicate that, under conditions where nuclear DNA synthesis is apparently unperturbed, the intracellular distribution of the deoxyribonucleotide biosynthetic enzymes studied is the same as that of glucose-6-phosphate dehydrogenase, a typical cytosol enzyme, and clearly differs from that of NMN adenylyltransferase, a nuclear enzyme.     

Covalent binding of 3-pyridinealdehyde nicotinamide adenine dinucleotide and substrate to glyceraldehyde 3-phosphate dehydrogenase.

Glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde-3-phoshate:nicotinamide adenine dinucleotide oxidoreductase (phosphorylating), EC 1.2.1.12) forms a complex with 3-pyridinealdehyde-NAD which survives precipitation with 7% perchloric acid. The molar ratio bound 3-pyridinealdehyde-NAD to the enzyme is 2.5 to 2.9. Lactate, malate, and alcohol dehydrogenases do not form acid-precipitable complexes with 3-pyridinealdehyde-NAD. 3-Pyridinealdehyde-deamino-NAD or glyceraldehyde 3-phosphate also forms an acid-stable complex with glyceraldehyde 3-phosphate dehydrogenase; however, NAD, 3-acetylpyridine-NAD, or thionicotinamide-NAD does not produce an acid-stable complex. Incubation of the glyceraldehyde 3-phosphate dehydrogenase with glyceraldehyde 3-phosphate, acetyl phosphate, iodoacetic acid, or iodosobenzoate inhibits the formation of the acid-stable complex with 3-pyridinealdehyde-NAD. Glyceraldehyde 3-phosphate or 3-pyridinealdehyde-NAD also prevents carboxymethylation of the active site cysteine-149 by[14-C]iodoacetic acid. These studies indicate that the aldehyde group of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of 3-pyridinealdehyde-NAD forms a thiohemiacetal linkage with cysteine-149 which is the substrate binding site for the dehydrogenase reaction. These findings may account for the fact that 3-pyridinealdehyde-NAD strongly inhibits the dehydrogenase and esterase activities of glyceraldehyde 3-phosphate dehydrogenase which require reduced cysteine-149. However, the analogue does not inhibit the acetyl phosphates activity of the enzyme for which the active site sulfhydryl residues must be oxidized.     

Inhibition of thymidylate synthase by pyridoxal phosphate

1. Pyridoxal phosphate (PLP) reversibly inhibited thymidylate synthase from Lactobacillus casei with a KI of 0.6-0.9 microM. 2. The inhibition was competitive with dUMP and noncompetitive with 5,10-methylenetetrahydrofolate which is consistent with an ordered addition of substrates. 3. The spectrum of PLP was altered by the addition of thymidylate synthase. The spectral changes suggest formation of a thiohemiacetal with an enzyme sulfhydryl group rather than Schiff base formation with a lysine side chain.     

Construction and expression of mouse thymidylate synthase minigenes

Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.     

Thymidylate synthase inhibition in cells with arrested DNA synthesis is not due to an allosteric interaction in the replitase complex

Activity of thymidylate synthase was measured in situ in leukemia cells by tritium release from [5-3H]dUrd. Aphidicolin, an inhibitor of DNA polymerase alpha, but not thymidylate synthase, caused a time dependent inhibition of the enzyme when added to the cells after [5-3H]dUrd. Cells treated with hydroxyurea and aphidicolin in sequence before addition of [5-3H]dUrd had a high initial thymidylate synthase activity that decreased with time. This pattern indicates that thymidylate synthase activity is linked to DNA synthesis; however, its inhibition by drugs that inhibit DNA synthesis may be due to accumulation of thymidine nucleotide(s), rather than to an allosteric interaction in the replitase complex.     

Isolation and functional effects of monoclonal antibodies binding to thymidylate synthase

Monoclonal antibodies against electrophoretically pure thymidylate synthase from HeLa cells have been produced. Antibodies (M-TS-4 and M-TS-9) from hybridoma clones were shown by enzyme-linked immunoassay to recognize thymidylate synthase from a variety of human cell lines, but they did not bind to thymidylate synthase from mouse cell lines. The strongest binding of antibodies was observed to enzyme from HeLa cells. These two monoclonal antibodies bind simultaneously to different antigenic sites on thymidylate synthase purified from HeLa cells, as reflected by a high additivity index and results of cross-linked radioimmunoassay. Both monoclonal antibodies inhibit the activity of thymidylate synthase from human cell lines. The strongest inhibition was observed with thymidylate synthase from HeLa cells. Monoclonal antibody M-TS-9 (IgM subclass) decreased the rate of binding of [3H]FdUMP to thymidylate synthase in the presence of 5,10-methylenetetrahydrofolate while M-TS-4 (IgG1) did not change the rate of ternary complex formation. These data indicate that the antibodies recognize different epitopes on the enzyme molecule.     

Regulation of thymidylate synthase gene expression in mouse fibroblasts synchronized by mitotic selection

Previous studies have shown that thymidylate synthase gene expression is regulated over a wide range in response to growth stimulation in cultured mouse fibroblasts. In the present study we show that the gene is also regulated during the cell cycle in continuously growing cells. Our analyses were conducted with a fluorodeoxyuridine-resistant mouse 3T6 cell line that overproduces thymidylate synthase and its mRNA by a factor of 50 due to gene amplification. Cells were synchronized by mitotic selection. RNA blot analyses showed that the amount of thymidylate synthase mRNA increased 5- to 10-fold as cells progressed from G1 through the middle of S phase. S1 nuclease protection assays showed that the pattern of 5' termini of thymidylate synthase mRNA was the same in G1 and S phase. Despite the large increase in thymidylate synthase mRNA content, the level of the enzyme increased only by a factor of 2 as cells progressed from G1 to mid S phase. This apparent discrepancy can be explained by the fact that the enzyme is highly stable.     

Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme

We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C. 2.1.1.45). The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons. The predicted amino acid sequence is 90% identical with that of the human enzyme. The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date. The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid. The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.      

Thermodynamic analysis of the emergence of new regulatory properties in a phosphoribulokinase-glyceraldehyde 3-phosphate dehydrogenase complex

Glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase exist as stable enzymes and as part of a complex in Chlamydomonas reinhardtii. We show here that phosphoribulokinase exerts an imprinting on glyceraldehyde 3-phosphate dehydrogenase, which affects its catalysis by decreasing the energy barrier of the reactions with NADH or NADPH by 3.8 +/- 0.5 and 1.3 +/- 0.3 kJ.mol(-1). Phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase within the complex are regulated by NADP(H) but not by NAD(H). The activities of the metastable phosphoribulokinase and glyceraldehyde 3-phosphate dehydrogenase released from the complex preincubated with NADP(H) are different from those of the metastable enzymes released from the untreated complex. NADP(H) increases phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase activities with a (~)K(0.5 (NADP)) of 0.68 +/- 0.16 mm and a (~)K(0.5 (NADPH)) of 2.93 +/- 0.87 mm and decreases NADH-dependent activity. 1 mm NADP increases the energy barrier of the NADH-glyceraldehyde 3-phosphate dehydrogenase-dependent reaction by 1.8 +/- 0.2 kJ.mol(-1) and decreases that of the reactions catalyzed by phosphoribulokinase and NADPH-glyceraldehyde 3-phosphate dehydrogenase by 3 +/- 0.2 and 1.2 +/- 0.3 kJ.mol(-1), respectively. These cofactors have no effect on the independent stable enzymes. Therefore, protein-protein interactions may give rise to new regulatory properties.     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.     

In situ autoradiographic detection of folylpolyglutamate synthetase activity

The enzyme folylpolyglutamate synthetase (FPGS) catalyzes the conversion of folate (pteroylmonoglutamate) to the polyglutamate forms (pteroylpolyglutamates) that are required for folate retention by mammalian cells. A rapid in situ autoradiographic assay for FPGS was developed which is based on the folate cofactor requirement of thymidylate synthase. Chinese hamster AUX B1 mutant cells lack FPGS activity and are unable to accumulate folate. As a result, the conversion of [6-3H]deoxyuridine to thymidine via the thymidylate synthase reaction is impaired in AUX B1 cells and no detectable label is incorporated into DNA. In contrast, FPGS in wild-type Chinese hamster CHO cells causes folate retention and enables the incorporation of [6-3H]deoxyuridine into DNA. Incorporation may be detected by autoradiography of monolayer cultures or of colonies replica plated onto polyester discs. Introduction of Escherichia coli FPGS into AUX B1 cells restores the activity of the thymidylate synthase pathway and demonstrates that the E. coli FPGS enzyme can provide pteroylpolyglutamates which function in mammalian cells.     

Inactivation of thymidylate synthase by chlorine disinfectants

The effects of two chlorine disinfectants, calcium hypochlorite (HTH) and 3-chloro-4,4-dimethyl-2-oxazolidinone (Agent I), on the activity of thymidylate synthase have been investigated. Although both disinfectants inactivated the enzyme, the following differences were observed: When the two disinfectants were used at the same total chlorine concentration, the rate and extent of inactivation were greater with Agent I than HTH. The substrate dUMP partially protected thymidylate synthase from inactivation by Agent I, but it did not appreciably protect against inactivation by HTH. Large changes in the ultraviolet spectrum of the enzyme occurred when it was treated with HTH, which suggests reactions with aromatic amino acid side chains; no spectral changes occurred when thymidylate synthase was treated with Agent I. Blocking the sulfhydryl groups of thymidylate synthase with sulfhydryl reagents prevented the irreversible inactivation of the enzyme by Agent I, but not by HTH.     

Induction, by thymidylate stress, of genetic recombination as evidenced by deletion of a transferred genetic marker in mouse FM3A cells.

Studies were made on the genetic consequences of methotrexate-directed thymidylate stress, focusing attention on a human thymidylate synthase gene that was introduced as a heterologous genetic marker into mouse thymidylate synthase-negative mutant cells. Thymidylate stress induced thymidylate synthase-negative segregants with concomitant loss of human thymidylate synthase activity with frequencies 1 to 2 orders of magnitude higher than the uninduced spontaneous level in some but not all transformant lines. Induction of the segregants was suppressed almost completely by cycloheximide and partially by caffeine. Thymidylate stress did not, however, induce mutations, as determined by measuring resistance to ouabain or 6-thioguanine. Thymidylate synthase-negative segregants were also induced by other means such as bromodeoxyuridine treatment and X-ray irradiation. In each of the synthase-negative segregants induced by thymidylate stress, a DNA segment including almost the whole coding region of the transferred human thymidylate synthase gene was deleted in a very specific manner, as shown by Southern blot analysis with a human Alu sequence and a human thymidylate synthase cDNA as probes. In the segregants that emerged spontaneously at low frequency, the entire transferred genetic marker was lost. In the segregants induced by X-ray irradiation, structural alterations of the genetic marker were random. These results show that thymidylate stress is a physiological factor that provokes the instability of this exogenously incorporated DNA in some specific manner and produces nonrandom genetic recombination in mammalian cells.     

Cloning, characterization and expression analysis of nucleotide metabolism-related genes of mycobacteriophage L5

The genomes of mycobacteriophages of the L5 family, which includes the lytic phage D29, contain several genes putatively linked to nucleotide-metabolizing functions. Two such genes, 48 and 50 , encoding thymidylate synthase and ribonucleotide reductase (RNR), respectively, were overexpressed in Escherichia coli and the recombinant proteins were biochemically characterized. It was established that Gp50 was a class II RNR having properties similar to that of the corresponding enzyme from Lactobacillus leichmanni , whereas Gp48 was a flavin-dependent thymidylate synthase (ThyX) that resembled the Paramecium bursaria chlorella virus-1 ThyX enzyme in its properties. That both these proteins play a role in phage development was evident from the observation that they were detectable soon after the lytic phase of growth commenced. Gp48 and 50 were also found to coimmunoprecipitate, which indicates the possible existence of an L5 thymidylate synthase complex. Thymidylate synthase assays revealed that during the intracellular stage of phage growth, a significant decrease in the host thymidylate synthase (ThyA) activity occurred. It appears that synthesis of the viral enzyme (ThyX) is necessary to compensate for this loss in activity. In general, the results suggest that phage-encoded nucleotide metabolism-related functions play an important role in the lytic propagation of L5 and related mycobacteriophages.     

Evidence for a substrate assisted conformational transformation of glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase exhibits half-site reactivity, the structural origin of which is obscure. Thermal inactivation kinetics, employed here as a probe for site-site heterogeneity in solution, show that green gram glyceraldehyde 3-phosphate dehydrogenase (in the absence and presence of phosphate and NAD+) loses activity in two distinct phases, each of which accounts for half of the initial activity. In the presence of substrate, glyceraldehyde 3-phosphate the relative amplitude of the slow phase increases, and at 0.06 mM glyceraldehyde 3-phosphate the time-course of inactivation corresponds to a single exponential decay. The data are consistent with a suggestion that glyceraldehyde 3-phosphate dehydrogenase may exist in two interconvertible conformations of different symmetry characteristics (C2 in equilibrium D2). The lower symmetry conformation (C2) predominates in the apoenzyme and in the presence of phosphate and NAD+. The higher symmetry conformation (D2) is stabilised by glyceraldehyde 3-phosphate.     

Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates. Experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamylates in antifolate action in cells

Folate analogs that inhibit dihydrofolate reductase result in only partial interconversion of tetrahydrofolate cofactors to dihydrofolate with preservation of the major portion of reduced cellular folate cofactors in L1210 leukemia cells. One possible explanation for this phenomenon is that low levels of dihydrofolate polyglutamates that accumulate in the presence of antifolates block thymidylate synthase to prevent depletion of reduced folate pools. This paper correlates biochemical analyses of rapid interconversions of radiolabeled folates and changes in purine and pyrimidine biosynthesis in L1210 murine leukemia cells exposed to antifolates with network thermodynamic computer modeling to assess this hypothesis. When cells are exposed to 1 microM trimetrexate there is an almost instantaneous inhibition of [3H] deoxyuridine or [14C]formate incorporation into nucleotides which is maximal within 5 min. This is associated with a rapid rise in cellular dihydrofolate (t1/2 approximately 1.5 min), which reaches a steady state that represents only 27.9% of the total folate pool. Pretreatment of cells with fluorodeoxyuridine, to inhibit thymidylate synthase by about 95% followed by trimetrexate only slows the rate of folate interconversion (t1/2 approximately 25 min) but not the final dihydrofolate level achieved. This is consistent with computer simulations which predict that direct inhibition of thymidylate synthase by 97, 98, and 99% should increase the half-time of dihydrofolate rise after trimetrexate to 40, 60, and 124 min, respectively, but the final level achieved is always the same as in cells with normal thymidylate synthase activity. The data reflect the high degree of catalytic activity of thymidylate synthase relative to tetrahydrofolate cofactor pools in the cells and the enormous extent of inhibition of this enzyme that is necessary to slow the rate of folate interconversions after addition of antifolates. The model predicts, and the data demonstrate, that virtually any residual thymidylate synthase activity will permit the interconversion of all tetrahydrofolate cofactors available for oxidation to dihydrofolate when dihydrofolate reductase activity is abolished, but the rate of interconversion will be slowed. Additional simulations indicate that the time course of cessation of tetrahydrofolate-dependent purine and pyrimidine biosynthesis after antifolates in these cells can be accounted for solely on the basis of tetrahydrofolate cofactor depletion alone. These data exclude the possibility that direct inhibition of thymidylate synthase by dihydrofolate polyglutamates, or any other intracellular folates that accumulate in cells after antifolates, can account for the rapid but partial interconversion of reduced folate cofactors to dihydrofolate.(ABSTRACT TRUNCATED AT 400 WORDS)