The structural mechanism for half-the-sites reactivity in an enzyme, thymidylate synthase, involves a relay of changes between subunits.

Thymidylate synthase (TS), a half-the-sites reactive enzyme, catalyzes the final step in the de novo biosynthesis of deoxythymidine monophosphate, dTMP, required for DNA replication. The cocrystal structure of TS from Pneumocystis carinii (PcTS), a new drug target for an important pathogen, with its substrate, deoxyuridine monophosphate (dUMP), and a cofactor mimic, CB3717, was determined. The structure, solved at 2.6 A resolution, shows an asymmetric dimer with two molecules of the substrate dUMP bound yet only one molecule of cofactor analogue bound. The structural evidence reveals that upon binding cofactor analogue and forming a covalent bond from the nucleophilic cysteine to the substrate, dUMP, at one active site, PcTS undergoes a conformational change that renders the opposite monomer incapable of forming a covalent bond or binding a molecule of cofactor analogue. The communication pathway between the two active sites is evident, allowing a structural definition of the basis of half-the-sites reactivity for thymidylate synthase and providing an example of such a mechanism for other half-the-sites reactive enzymes.     

Mechanistic and structural basis for inhibition of thymidylate synthase ThyX

Nature has established two mechanistically and structurally unrelated families of thymidylate synthases that produce de novo thymidylate or dTMP, an essential DNA precursor. Representatives of the alternative flavin-dependent thymidylate synthase family, ThyX, are found in a large number of microbial genomes, but are absent in humans. We have exploited the nucleotide binding pocket of ThyX proteins to identify non-substrate-based tight-binding ThyX inhibitors that inhibited growth of genetically modified Escherichia coli cells dependent on thyX in a manner mimicking a genetic knockout of thymidylate synthase. We also solved the crystal structure of a viral ThyX bound to 2-hydroxy-3-(4-methoxybenzyl)-1,4-naphthoquinone at a resolution of 2.6 Å. This inhibitor was found to bind within the conserved active site of the tetrameric ThyX enzyme, at the interface of two monomers, partially overlapping with the dUMP binding pocket. Our studies provide new chemical tools for investigating the ThyX reaction mechanism and establish a novel mechanistic and structural basis for inhibition of thymidylate synthesis. As essential ThyX proteins are found e.g. in Mycobacterium tuberculosis and Helicobacter pylori, our studies have also potential to pave the way towards the development of new anti-microbial compounds.     

Evolutionarily conserved structural and functional roles of the FYVE domain

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.     

Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX

By using biochemical and structural analyses, we have investigated the catalytic mechanism of the recently discovered flavin-dependent thymidylate synthase ThyX from Paramecium bursaria chlorella virus-1 (PBCV-1). Site-directed mutagenesis experiments have identified several residues implicated in either NADPH oxidation or deprotonation activity of PBCV-1 ThyX. Chemical modification by diethyl pyrocarbonate and mass spectroscopic analyses identified a histidine residue (His53) crucial for NADPH oxidation and located in the vicinity of the redox active N-5 atom of the FAD ring system. Moreover, we observed that the conformation of active site key residues of PBCV-1 ThyX differs from earlier reported ThyX structures, suggesting structural changes during catalysis. Steady-state kinetic analyses support a reaction mechanism where ThyX catalysis proceeds via formation of distinct ternary complexes without formation of a methyl enzyme intermediate.     

Variants of human thymidylate synthase with loop 181–197 stabilized in the inactive conformation

Loop 181–197 of human thymidylate synthase (hTS) populates two major conformations, essentially corresponding to the loop flipped by 180°. In one of the conformations, the catalytic Cys195 residue lies distant from the active site making the enzyme inactive. Ligands stabilizing this inactive conformation may function as allosteric inhibitors. To facilitate the search for such inhibitors, we have expressed and characterized several mutants designed to shift the equilibrium toward the inactive conformer. In most cases, the catalytic efficiency of the mutants was only somewhat impaired with values of k_cat/K_m reduced by factors in a 2–12 range. One of the mutants, M190K, is however unique in having the value of k_cat/K_m smaller by a factor of ～7500 than the wild type. The crystal structure of this mutant is similar to that of the wt hTS with loop 181–197 in the inactive conformation. However, the direct vicinity of the mutation, residues 188–194 of this loop, assumes a different conformation with the positions of C_α shifted up to 7.2 Å. This affects region 116–128, which became ordered in M190K while it is disordered in wt. The conformation of 116–128 is however different than that observed in hTS in the active conformation. The side chain of Lys190 does not form contacts and is in solvent region. The very low activity of M190K as compared to another mutant with a charged residue in this position, M190E, suggests that the protein is trapped in an inactive state that does not equilibrate easily with the active conformer.     

The identification of thymidylate synthase peptide domains located in the interface region that bind thymidylate synthase mRNA

Thymidylate synthase (TS) is a critical chemotherapeutic target and intracellular levels of TS are an important determinant of sensitivity to TS inhibitors. Translational autoregulation represents one cellular mechanism for controlling the level of expression of TS. This mechanism involves the binding of TS protein to its own messenger RNA (mRNA), thus, repressing translational efficiency. The presence of excess substrate or inhibitors of TS leads to derepression of protein binding to mRNA, resulting in increased translational efficiency and ultimately increased levels of TS protein. TS protein has been shown to bind to two distinct areas on its mRNA. The goal of the present work is to define the TS domains responsible for this interaction. Using a separate series of overlapping 17-mer peptides spanning the length of both the human and Escherichia coli TS sequences, we have identified six potential domains located in the interface region of the TS protein that bind TS mRNA. The identified domains that bind TS mRNA include three concordant regions in both the human and E. coli peptide series. Five of the six binding peptides contain at least one invariant arginine residue, which has been shown to be critical in other well-defined protein-RNA interactions. These data suggest that the identified highly conserved protein domains, which occur at the homodimeric interface of TS, represent potential participating sites for binding of TS protein to its mRNA.     

Human thymidylate synthase with loop 181-197 stabilized in an inactive conformation: ligand interactions, phosphorylation, and inhibition profiles

Thymidylate synthase (TS) is a well-validated cancer target that undergoes conformational switching between active and inactive states. Two mutant human TS (hTS) proteins are predicted from crystal structures to be stabilized in an inactive conformation to differing extents, with M190K populating the inactive conformation to a greater extent than A191K. Studies of intrinsic fluorescence and circular dichroism revealed that the structures of the mutants differ from those of hTS. Inclusion of the substrate dUMP was without effect on M190K but induced structural changes in A191K that are unique, relative to hTS. The effect of strong stabilization in an inactive conformation on protein phosphorylation by casein kinase 2 (CK2) was investigated. M190K was highly phosphorylated by CK2 relative to an active-stabilized mutant, R163K hTS. dUMP had no detectable effect on phosphorylation of M190K; however, dUMP inhibited phosphorylation of hTS and R163K. Studies of temperature dependence of catalysis revealed that the E(act) and temperature optimum are higher for A191K than hTS. The potency of the active-site inhibitor, raltitrexed, was lower for A191K than hTS. The response of A191K to the allosteric inhibitor, propylene diphosphonate (PDPA) was concentration dependent. Mixed inhibition was observed at low concentrations; at higher concentrations, A191K exhibited nonhyperbolic behavior with respect to dUMP and inhibition of catalysis was reversed by substrate saturation. In summary, inactive-stabilized mutants differ from hTS in thermal stability and response to substrates and PDPA. Importantly, phosphorylation of hTS by CK2 is selective for the inactive conformation, providing the first indication of physiological relevance for conformational switching.     

Biotin synthase mechanism: mutagenesis of the YNHNLD conserved motif

Biotin synthase, a member of the "radical SAM" family, catalyzes the final step of the biotin biosynthetic pathway, namely, the insertion of a sulfur atom into dethiobiotin (DTB). The active form of the enzyme contains two iron-sulfur clusters, a [4Fe-4S](2+) cluster liganded by Cys-53, Cys-57, and Cys-60 and the S-adenosylmethionine (AdoMet or SAM) cosubstrate and a [2Fe-2S](2+) cluster liganded by Cys-97, Cys-128, Cys-188, and Arg-260. Single-point mutation of each of these six conserved cysteines produced inactive variants. In this work, mutants of other highly conserved residues from the Y(150)NHNLD motif are described. They have properties similar to those of the wild-type enzyme with respect to their cluster content and characteristics. For all of them, the as-isolated form, which contains an air-stable [2Fe-2S](2+) center, can additionally accommodate an air-sensitive [4Fe-4S](2+) center which is generated by incubation under anaerobic conditions with Fe(2+) and S(2-). Their spectroscopic properties are similar to those of the wild type. However, they are inactive, except the mutant H152A that exhibits a weak activity. We show that the mutants, inactive in producing biotin, are also unable to cleave AdoMet and to produce the deoxyadenosyl radical (AdoCH(2)(*)). In the case of H152A, a value of 5.5 +/- 0.4 is found for the 5'-deoxyadenosine (AdoCH(3)):biotin ratio, much higher than the value of 2.8 +/- 0.3 usually observed with the wild type. This reveals a greater contribution of the abortive process in which the AdoCH(2)(*) radical is quenched by hydrogen atoms from the protein or from some components of the system. Thus, in this case, the coupling between the production of AdoCH(2)(*) and its reaction with the hydrogen at C-6 and C-9 of DTB is less efficient than that in the wild type, probably because of geometry's perturbation within the active site.     

The mechanism and structure of thymidylate synthetase

We have been involved in studies of the mechanism, inhibition and structure of the enzyme thymidylate synthetase. Knowledge of fundamental catalytic features of thymidylate synthetase has accumulated over the past decade, and will be described. Recently, we have been involved in studies of the x-ray crystallography of thymidylate synthetase, the first phase of which has been completed.     

Catalytic residues Lys197 and Arg199 of Bacillus subtilis phosphoribosyl diphosphate synthase. Alanine-scanning mutagenesis of the flexible catalytic loop

Eleven of the codons specifying the amino acids of the flexible catalytic loop [KRRPRPNVAEVM(197-208)] of Bacillus subtilis phosphoribosyl diphosphate synthase have been changed individually to specify alanine. The resulting variant enzyme forms, as well as the wildtype enzyme, were produced in an Escherichia coli strain lacking endogenous phosphoribosyl diphosphate synthase activity and purified to near homogeneity. The B. subtilis phosphoribosyl diphosphate synthase mutant variants K197A and R199A were studied in detail. The physical properties of the two enzymes were similar to those of the wildtype enzyme. Kinetic characterization showed that the V(max) values of the K197A and R199A mutant enzymes were more than 30 000- and more than 24 000-fold reduced, respectively, compared to the wildtype enzyme. The K(m) values for ATP and ribose 5-phosphate of the two mutant enzymes were essentially unchanged. V(app) values of the remaining mutant enzymes were much less affected, ranging from 20 to 100% of the V(max) value of the wildtype enzyme. The data presented show that Lys197 and Arg199 are important in stabilization of the transition state.     

A quantum mechanics/molecular mechanics study of the catalytic mechanism of the thymidylate synthase

A theoretical study of the molecular mechanism of the thymidylate synthase-catalyzed reaction has been carried out using hybrid quantum mechanics/molecular mechanics methods. We have examined all of the stationary points (reactants, intermediates, transition structures, and products) on the multidimensional potential energy surfaces for the multistep enzymatic process. The characterization of these relevant structures facilitates the gaining of insight into the role of the different residues in the active site. Furthermore, analysis of the full energy profile has revealed that the step corresponding to the reduction of the exocyclic methylene intermediate by hydride transfer from the 6S position of 5,6,7,8-tetrahydrofolate (H4folate), forming dTMP and 7,8-dihydrofolate (H2folate), is the rate-limiting step, in accordance with the experimental data. In this step, the hydride transfer and the scission of an overall conserved active site cysteine residue (Cys146 in Escherichia coli) take place in a concerted but very asynchronous way. These findings have also been tested with primary and secondary deuterium, tritium, and sulfur kinetic isotope effects, and the calculations have been compared to experimental data. Finally, the incorporation of high-level quantum mechanical corrections to the semiempirical AM1 Hamiltonian into our hybrid scheme has allowed us to obtain reasonable values of the energy barrier for the rate-limiting step. The resulting picture of the complete multistep enzyme mechanism that is obtained reveals several new features of substantial mechanistic interest.     

Amino acid substitution analysis of E. coli thymidylate synthase: The study of a highly conserved region at the N-terminus

Amino acid substitution analysis within a highly conserved region of Escherichia coli thymidylate synthase (TS), using suppression of amber mutations by tRNA suppressors, has yielded a bank of 124 new mutationally altered TS proteins. These mutant proteins have been used to study the structure-function relationship of the Escherichia coli TS protein at the N-terminus corresponding to residues 20 through 35. This region contains a block of amino acids whose sequence has been well conserved among other known TS proteins from various organisms. Positions 20 through 25 contain a surface loop structure and positions 26 through 35 encompass a β-strand. We find that residues surrounding a β-bulge structure within the β-strand are particularly sensitive to amino acid substitution, suggesting that this structure is maintained by a highly ordered packing arrangement. Three residues in the surface loop that are present at the base of the substrate binding pocket are also sensitive to amino acid substitution. The remainder of the conserved sites, including those at the dimer interface, are tolerant to most, if not all, of the substitutions tested. © 1992 Wiley-Liss, Inc.     

The multiple roles of conserved arginine 286 of 1-aminocyclopropane-1-carboxylate synthase. Coenzyme binding, substrate binding, and beyond

A pyridoxal 5'-phosphate (PLP)-dependent enzyme, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (S-adenosyl-L-Met methylthioadenosine-lyase, EC 4.4.1.14), catalyzes the conversion of S-adenosyl-L-methionine (AdoMet) to ACC. A tomato ACC synthase isozyme (LE-ACS2) with a deletion of 46 amino acids at the C terminus was chosen as the control enzyme for the study of the function of R286 in ACC synthase. R286 of the tomato ACC synthase was mutated to a leucine via site-directed mutagenesis. The ACC synthase mutant R286L was purified using a simplified two-step purification protocol. Circular dichroism (CD) analysis indicated that the overall three-dimensional structure of the mutant was indistinguishable from that of the control enzyme. Fluorescence spectroscopy revealed that the binding affinity of R286L ACC synthase for its cofactor PLP was reduced 20- to 25-fold compared with control. Kinetic analysis of R286L showed that this mutant ACC synthase had a significantly reduced turnover number (k(cat)) of 8.2 x 10(-3) s(-1) and an increased K(m) of 730 microM for AdoMet, leading to an 8,000-fold decrease in overall catalytic efficiency compared with the control enzyme. Thus, R286 of tomato ACC synthase is involved in binding both PLP and AdoMet.     

Inhibition of thymidylate synthase by pergularinine, tylophorinidine and deoxytubulosine

The activity of thymidylate synthase (TS) purified in our laboratory from Lactobacillus leichmannii was inhibited by pergularinine (PGL) and tylophorinidine (TPD) and deoxytubulosine (DTB) isolated from the Indian medicinal plants Pergularia pallida and Alangium lamarckii respectively. Cytotoxicity studies showed that cell growth of L. leichmannii was inhibited (IC50 = 40-45 microM) by all the three alkaloids, the concentrations > 80-90 microM resulting in complete loss of the enzyme activity. Ki values of the enzyme calculated from Lineweaver-Burk and Dixon plots for PGL, TPD and DTB were 10 x 10(-6) M, 9 x 10(-6) M and 7 x 10(-6) M respectively. These are typed as 'non-competitive' inhibitors of TS. All the three alkaloids inhibited (IC50 = 50 microM) the elevated TS activity of leukocytes in cancer patients with clinically diagnosed chronic myelocytic leukemia (n = 10), acute lymphocytic leukemia (n = 8) and metastatic solid tumours (n = 3).     

Dissecting the roles of a strictly conserved tyrosine in substrate recognition and catalysis by pseudouridine 55 synthase

Sequence alignment of the TruA, TruB, RsuA, and RluA families of pseudouridine synthases (PsiS) identifies a strictly conserved aspartic acid, which has been shown to be the critical nucleophile for the PsiS-catalyzed formation of pseudouridine (Psi). However, superposition of the representative structures from these four families of enzymes identifies two additional amino acids, a lysine or an arginine (K/R) and a tyrosine (Y), from a K/RxY motif that are structurally conserved in the active site. We have created a series of Thermotoga maritima and Escherichia coli pseudouridine 55 synthase (Psi55S) mutants in which the conserved Y is mutated to other amino acids. A new crystal structure of the T. maritima Psi55S Y67F mutant in complex with a 5FU-RNA at 2.4 A resolution revealed formation of 5-fluoro-6-hydroxypseudouridine (5FhPsi), the same product previously seen in wild-type Psi55S-5FU-RNA complex structures. HPLC analysis confirmed efficient formation of 5FhPsi by both Psi55S Y67F and Y67L mutants but to a much lesser extent by the Y67A mutant when 5FU-RNA substrate was used. However, both HPLC analysis and a tritium release assay indicated that these mutants had no detectable enzymatic activity when the natural RNA substrate was used. The combined structural and mutational studies lead us to propose that the side chain of the conserved tyrosine in these four families of PsiS plays a dual role within the active site, maintaining the structural integrity of the active site through its hydrophobic phenyl ring and acting as a general base through its OH group for the proton abstraction required in the last step of PsiS-catalyzed formation of Psi.     

Polymorphism of the thymidylate synthase gene and thymidylate synthase levels in colon cancer cell lines and different tissues of colorectal cancer patients

In a panel of 18 colon cancer cell lines we found that the thymidylate synthase (TS) genotype was related to TS enzyme activity, but not to TS protein and mRNA levels. In addition, no relation with drug sensitivity was observed. TS genotyping of different tissues from 78 colorectal cancer patients revealed a high level of homology in polymorphic status between normal and malignant tissues and the heterozygous genotype to be the most frequent.     

Cloning, purification, and properties of Candida albicans thymidylate synthase.

The thymidylate synthase (TS) gene was isolated from a genomic Candida albicans library by functional complementation of a Saccharomyces cerevisiae strain deficient in TS. The gene was localized on a 4-kilobase HindIII DNA fragment and was shown to be expressed in a Thy- strain of Escherichia coli. The nucleotide sequence of the TS gene predicted a protein of 315 amino acids with a molecular weight of 36,027. The gene was cloned into a T7 expression vector in E. coli, allowing purification of large amounts of C. albicans TS. It was also purified from a wild-type C. albicans strain. Comparison of several enzyme properties including analysis of amino-terminal amino acid sequences showed the native and cloned C. albicans TS to be the same.     

Cofactor triggers the conformational change in thymidylate synthase: implications for an ordered binding mechanism.

We have solved crystal structures of two complexes with Escherichia coli thymidylate synthase (TS) bound either to the cofactor analog N10-propargyl-5,8-dideazafolate (CB3717) or to a tighter binding polygutamyl derivative of CB3717. These structures suggest that cofactor binding alone is sufficient to induce the conformational change in TS; dUMP binding is not required. Because polyglutamyl folates are the primary cofactor form in vivo, and because they can bind more tightly than dUMP to TS, these structures may represent a key intermediate along the TS reaction pathway. These structures further suggest that the dUMP binding site is accessible in the TS-cofactor analog binary complexes. Conformational flexibility of the binary complex may permit dUMP to enter the active site of TS while the cofactor is bound. Alternatively, dUMP may enter the active site from the opposite side that the cofactor appears to enter; that is, through a portal flanked by arginines that also coordinate the phosphate group in the active site. Entry of dUMP through this portal may allow dUMP to bind to a TS-cofactor binary complex in which the complex has completed its conformational transition to the catalytically competent structure.     

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. The known crystal structures for WT-TS, Y94F, and R126E, surprisingly, showed no structural differences that could explain the difference in their susceptibility to trypsin. One explanation is that the mutations could perturb the dynamic equilibrium of the dimeric state of the mutants as to increase their dissociation to monomers, which being less structured than the dimer, would be hydrolyzed more readily by trypsin. Earlier studies appear to support this proposal since conditions that promote subunit dissociation in solutions of R126E with other inactive mutants, such as dilution, low concentrations of urea, and elevated pH, greatly enhance the rate of restoration of TS activity. Analytic ultracentrifuge studies with various TSs in urea, or at pH 9.0, or that have been highly diluted are, for the most part, in agreement with this thesis, since these conditions are associated with an increase in dissociation to monomers, particularly with the mutant TSs. However, these studies do not rule out the possibility that conformation differences among the various TS dimers are responsible for the differences in susceptibility to trypsin, particularly at high concentrations of protein where the WT-TS and mutants are mainly dimers.