1 2009 Vol: 458(7240):919-923. DOI: 10.1038/nature07973

An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene

Biosynthesis of the DNA base thymine depends on activity of the enzyme thymidylate synthase to catalyse the methylation of the uracil moiety of 2′-deoxyuridine-5′-monophosphate. All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate1, 2. This functionality has been demonstrated for classical thymidylate synthases, including human thymidylate synthase, and is instrumental in mechanism-based inhibition of these enzymes. Here we report an example of thymidylate biosynthesis that occurs without an enzymatic nucleophile. This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3, 4, 5. Our findings indicate that the putative active site nucleophile is not required for FDTS catalysis, and no alternative nucleophilic residues capable of serving this function can be identified. Instead, our findings suggest that a hydride equivalent (that is, a proton and two electrons) is transferred from the reduced flavin cofactor directly to the uracil ring, followed by an isomerization of the intermediate to form the product, 2′-deoxythymidine-5′-monophosphate. These observations indicate a very different chemical cascade than that of classical thymidylate synthases or any other known biological methylation. The findings and chemical mechanism proposed here, together with available structural data, suggest that selective inhibition of FDTSs, with little effect on human thymine biosynthesis, should be feasible. Because several human pathogens depend on FDTS for DNA biosynthesis, its unique mechanism makes it an attractive target for antibiotic drugs.

Mentions
Figures
Figure 1: Thymidylate synthase mechanisms.a, The chemical mechanism for the classical thymidylate synthase catalysed reaction1, 2. b, The chemical mechanism for the FDTS proposed hitherto11. c, The newly proposed mechanism for the FDTS that does not rely on an enzymatic nucleophile. The conserved enzymatic nucleophile is orange, the methylene is purple, the reducing hydride from H4folate is green, and the hydride from FADH2 is red. R = 2'-deoxyribose-5'-phosphate; R' = (p-aminobenzoyl)-glutamate; R'' = adenosine-5'-pyroposphate-ribityl. Figure 2: Crystal structures of the FDTS–FAD–dUMP complex.a, Wild-type TmFDTS (Protein Data Bank 1o26); b, S88A mutant (Protein Data Bank 3g4a); c, S88C mutant (Protein Data Bank 3g4c). The distance between the C6 carbon of dUMP and the reducing centre of the flavin (N5 of FAD) is 3.4 Å for all three enzymes. The distances of the side chain of residue 88 to C6 are 4.3, 4.5 and 4.1 Å, for wild-type FDTS, S88A and S88C, respectively. The electron density maps are 2Fo - Fc with a contour level of 1.0 sigma. Figure 3: Hydride flow.An illustration of two experimental approaches to examine the hydride flow in the reaction catalysed by the thermophilic TmFDTS at reduced temperature (37 °C). Experiment A was performed in a D2O buffer using 6H-dUMP (that is, unlabelled dUMP). Experiment B was performed in an H2O buffer using 6D-dUMP (see Supplementary Information). Percentages below each species represent the relative quantities of product formation as indicated by 1H and 2H NMR.
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References
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    • . . . All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate1, 2 . . .
    • . . . The catalytic mechanism of classical thymidylate synthases is presented in Fig. 1a1, 2 . . .
    • . . . a, The chemical mechanism for the classical thymidylate synthase catalysed reaction1, 2. b, The chemical mechanism for the FDTS proposed hitherto11. c, The newly proposed mechanism for the FDTS that does not rely on an enzymatic nucleophile . . .
    • . . . Because the 5H position is always replaced during the synthesis of dTMP1, 2, 4 its isotopic labelling can be disregarded. . . .
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    • . . . All known thymidylate synthases rely on an active site residue of the enzyme to activate 2′-deoxyuridine-5′-monophosphate1, 2 . . .
    • . . . The catalytic mechanism of classical thymidylate synthases is presented in Fig. 1a1, 2 . . .
    • . . . a, The chemical mechanism for the classical thymidylate synthase catalysed reaction1, 2. b, The chemical mechanism for the FDTS proposed hitherto11. c, The newly proposed mechanism for the FDTS that does not rely on an enzymatic nucleophile . . .
    • . . . Another test for similar Michael addition to the C6 of dUMP is the dehalogenation of 5-bromo-dUMP (5Br-dUMP)2, 16 . . .
    • . . . Because the 5H position is always replaced during the synthesis of dTMP1, 2, 4 its isotopic labelling can be disregarded. . . .
  3. Myllykallio, H. et al. An alternative flavin-dependent mechanism of thymidylate synthesis. Science 297, 105-107 , .
    • . . . This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3, 4, 5 . . .
    • . . . A recently discovered class of thymidylate synthases, the FDTSs3, 6, 7, is encoded by the thyX gene and has been found primarily in prokaryotes and viruses3, 8, including several pathogens and biological warfare agents (see http://www.cdc.gov) . . .
  4. Leduc, D. et al. Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers. Proc. Natl Acad. Sci. USA 101, 7252-7257 , .
    • . . . This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3, 4, 5 . . .
    • . . . Although both mutations were found to retain activity4, it was assumed that an adjacent serine (Ser 85) could have rescued the activity of S84A . . .
    • . . . These results were used to propose that Ser 84 activates dUMP (Fig. 1b)4, 11. . . .
    • . . . Nevertheless, a MALDI–TOF analysis of the trypsin-digested S88C TmFDTS indicated that Cys 88 is bound to dUMP (see Supplementary Information), as previously reported for HpFDTS4 . . .
    • . . . In the past, we and others4, 11 suggested that these findings support the mechanism illustrated in Fig. 1b, but the current findings contradict that mechanism and required further tests . . .
    • . . . Because the 5H position is always replaced during the synthesis of dTMP1, 2, 4 its isotopic labelling can be disregarded. . . .
  5. Chernyshev, A., Fleischmann, T. & Kohen, A. Thymidyl biosynthesis enzymes as antibiotic targets. Appl. Microbiol. Biotechnol. 74, 282-289 , .
    • . . . This unusual biosynthetic pathway occurs in organisms containing the thyX gene, which codes for a flavin-dependent thymidylate synthase (FDTS), and is present in several human pathogens3, 4, 5 . . .
    • . . . FDTSs share no structure or sequence homology with classical thymidylate synthases, and thus present a promising new frontier for antibacterial/antiviral drug development5, 6, 7. . . .
    • . . . Notably, such a chemical mechanism is very different from that of classical thymidylate synthases, and along with structural differences, may help explain why classical thymidylate synthase inhibitors have a reduced effect on FDTSs5 . . .
  6. Lesley, S. A. et al. Structural genomics of the Thermotoga maritima proteome implemented in a high-throughput structure determination pipeline. Proc. Natl Acad. Sci. USA 99, 11664-11669 , .
    • . . . A recently discovered class of thymidylate synthases, the FDTSs3, 6, 7, is encoded by the thyX gene and has been found primarily in prokaryotes and viruses3, 8, including several pathogens and biological warfare agents (see http://www.cdc.gov) . . .
    • . . . FDTSs share no structure or sequence homology with classical thymidylate synthases, and thus present a promising new frontier for antibacterial/antiviral drug development5, 6, 7. . . .
    • . . . The active site of this enzyme contains a strictly conserved serine, Ser 88, and no alternative nucleophilic residues (see refs 6, 7 and Supplementary Information) . . .
    • . . . The FDTS from T. maritima (TM0449, GenBank accession number NP228259), and its mutants S88A and S88C, were expressed and purified as previously described6 . . .
    • . . . The FDTS from T. maritima (TM0449, GenBank accession number NP228259) and its mutants S88A and S88C were expressed and purified as previously described6 . . .
  7. Mathews, I. I. et al. Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein. Structure 11, 677-690 , .
    • . . . A recently discovered class of thymidylate synthases, the FDTSs3, 6, 7, is encoded by the thyX gene and has been found primarily in prokaryotes and viruses3, 8, including several pathogens and biological warfare agents (see http://www.cdc.gov) . . .
    • . . . Crystal structures of FDTSs from three very different organisms7, 12, 13 placed this conserved serine about 4 Å from the C6 position of dUMP (for example, Fig. 2a) . . .
    • . . . The active site of this enzyme contains a strictly conserved serine, Ser 88, and no alternative nucleophilic residues (see refs 6, 7 and Supplementary Information) . . .
    • . . . In contrast to classical thymidylate synthase, FDTS does not covalently bind 5F-dUMP upon incubation with CH2H4folate, as confirmed by both MALDI–TOF analysis (see Supplementary Information) and crystal structure analysis (Protein Data Bank 1o28, ref. 7) obtained under similar conditions . . .
  8. Leduc, D. et al. Two distinct pathways for thymidylate (dTMP) synthesis in (hyper)thermophilic Bacteria and Archaea. Biochem. Soc. Trans. 32, 231-235 , .
    • . . . A recently discovered class of thymidylate synthases, the FDTSs3, 6, 7, is encoded by the thyX gene and has been found primarily in prokaryotes and viruses3, 8, including several pathogens and biological warfare agents (see http://www.cdc.gov) . . .
  9. Escartin, F., Skouloubris, S., Liebl, U. & Myllykallio, H. Flavin-dependent thymidylate synthase X limits chromosomal DNA replication. Proc. Natl Acad. Sci. USA 105, 9948-9952 , .
    • . . . It has recently been suggested that thyX limits chromosomal replication in these organisms9 . . .
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    • . . . This results in the production of H4folate rather than H2folate10, 11 . . .
  11. Agrawal, N., Lesley, S. A., Kuhn, P. & Kohen, A. Mechanistic studies of a flavin-dependent thymidylate synthase. Biochemistry 43, 10295-10301 , .
    • . . . a, The chemical mechanism for the classical thymidylate synthase catalysed reaction1, 2. b, The chemical mechanism for the FDTS proposed hitherto11. c, The newly proposed mechanism for the FDTS that does not rely on an enzymatic nucleophile . . .
    • . . . This results in the production of H4folate rather than H2folate10, 11 . . .
    • . . . These results were used to propose that Ser 84 activates dUMP (Fig. 1b)4, 11. . . .
    • . . . Activity tests for S88A and S88C (for details see ref. 11 and Supplementary Information) indicated that both mutants were still active . . .
    • . . . We have previously found that when conducting the FDTS reaction in D2O (50% D), deuteration of the reduced flavin leads to deuterated dTMP (using electron spray ionization mass spectrometry, ESI-MS, analysis), and that reaction with tritiated 6T-CH2H4folate yields 6T-H4folate11 . . .
    • . . . In the past, we and others4, 11 suggested that these findings support the mechanism illustrated in Fig. 1b, but the current findings contradict that mechanism and required further tests . . .
    • . . . Lack of stereoselectivity at reduced temperature has already been observed during the reductive-half reaction of FDTS, which transfers both 4-(R) and 4-(S) hydride of NADPH11 . . .
    • . . . The activities of these enzymes were determined using a [2-14C]dUMP assay which is a modification of the procedure developed and described in ref. 11 . . .
    • . . . A Supelco reverse phase column (Discover series 250 mm × 4.6 mm) was used starting with 100 mM KH2PO4 (pH 6.0) followed by a methanol gradient as described elsewhere11 . . .
    • . . . The activity assay ([2-14C]dUMP assay) was a modification of the procedure developed and described in ref. 11 . . .
  12. Graziani, S. et al. Catalytic mechanism and structure of viral flavin-dependent thymidylate synthase ThyX. J. Biol. Chem. 281, 24048-24057 , .
    • . . . Crystal structures of FDTSs from three very different organisms7, 12, 13 placed this conserved serine about 4 Å from the C6 position of dUMP (for example, Fig. 2a) . . .
  13. Sampathkumar, P. et al. Structure of the Mycobacterium tuberculosis flavin dependent thymidylate synthase (MtbThyX) at 2.0 Å resolution. J. Mol. Biol. 352, 1091-1104 , .
    • . . . Crystal structures of FDTSs from three very different organisms7, 12, 13 placed this conserved serine about 4 Å from the C6 position of dUMP (for example, Fig. 2a) . . .
    • . . . We also solved the crystal structures of TmFDTS with FAD and both 5-halogenated-dUMPs (Protein Data Bank 1o27 and 1o28); the 5Br-dUMP–FAD structure for FDTS from Mycobacterium tuberculosis (Protein Data Bank 2af6) was solved by ref. 13 . . .
  14. Hong, B., Maley, F. & Kohen, A. The role of Y94 in proton and hydride transfers catalyzed by thymidylate synthase. Biochemistry 46, 14188-14197 , .
    • . . . Even in solution, cysteine covalently binds to the C6 position of uracil14 . . .
    • . . . A method to substitute the 5D into 5H without affecting 6D has been developed14; however, such substitution was not used in the current preparation because the thymidylate synthase reaction is a substitution reaction where a methyl group replaces the 5H of dUMP to form dTMP . . .
  15. Hyatt, D. C., Maley, F. & Montfort, W. R. Use of strain in a stereospecific catalytic mechanism: crystal structures of Escherichia coli thymidylate synthase bound to FdUMP and methylenetetrahydrofolate. Biochemistry 36, 4585-4594 , .
    • . . . A critical piece of evidence for the covalent bond between the active site cysteine in classical thymidylate synthase and dUMP is the crystal structure of a covalently bound 5-flouro-dUMP (5F-dUMP) in complex with CH2H4folate (Protein Data Bank accession 1tls, ref. 15) . . .
  16. Wataya, Y. & Santi, D. V. Thymidylate synthase catalyzed dehalogenation of 5-bromo-and 5-iodo-deoxyuridylate. Biochem. Biophys. Res. Commun. 67, 818-823 , .
    • . . . Another test for similar Michael addition to the C6 of dUMP is the dehalogenation of 5-bromo-dUMP (5Br-dUMP)2, 16 . . .
    • . . . The 5Br-dUMP assay was adopted from ref. 16 . . .
  17. Hong, B. & Kohen, A. Microscale synthesis of isotopically labeled 6R-N5, N10 methylene-5, 6, 7, 8-tetrahydrofolate. J. Labelled Comp. Radiopharm. 48, 759-769 , .
    • . . . These results contrast the same experiments with classical thymidylate synthases, where reactions performed in D2O do not incorporate deuterium into the dTMP and the labelled hydride from CH2H4folate always transfers to the dTMP17 . . .
  18. Gattis, S. G. & Palfey, B. A. Direct observation of the participation of flavin in product formation by thyX-encoded thymidylate synthase. J. Am. Chem. Soc. 127, 832-833 , .
    • . . . In Fig. 1c we propose a new chemical mechanism consistent with current data and previous findings18, wherein a hydride equivalent from the N5 of FADH2 is transferred to C6 of dUMP (Fig. 1c, step 1) . . .
  19. Klötzer, W. Two isomers of thymine. Monatsh. Chem. 104, 415-420 , .
    • . . . The intermediate proposed here is unique in nucleotide biochemistry, but this isomer of the thymine moiety is chemically feasible and quite stable in solution19 . . .
    • . . . Because the isomerization of the putative intermediate (Fig. 1c, step 4) does not occur rapidly in solution19, the enzyme could catalyse this transformation by the two mechanisms illustrated in Fig. 4 . . .
  20. Carey, F. A. & Sundberg, R. J. Advanced Organic Chemistry, Part A (Kluwer Academic/Plenum, 2000) , .
    • . . . Alternatively, the thermodynamic driving force (>6 kcal mol-1 as estimated from semi-empirical quantum mechanical calculations) could favour a 1,3-sigmatropic rearrangement (1,3-hydride shift)20 . . .
  21. Brown, B. J., Deng, Z., Karplus, P. A. & Massey, V. On the active site of Old Yellow Enzyme. Role of histidine 191 and asparagine 194. J. Biol. Chem. 273, 32753-32762 , .
    • . . . For example, dihydroorotate dehydrogenase and old yellow enzyme are other flavo-proteins that catalyse similar chemistry21, 22. . . .
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    • . . . For example, dihydroorotate dehydrogenase and old yellow enzyme are other flavo-proteins that catalyse similar chemistry21, 22. . . .
  23. Waldman, A. S., Haeusslein, E. & Milman, G. Purification and characterization of herpes simplex virus (type 1) thymidine kinase produced in Escherichia coli by a high efficiency expression plasmid utilizing a lambda PL promoter and cI857 temperature- sensitive repressor. J. Biol. Chem. 258, 11571-11575 , .
    • . . . Stroud’s laboratory at UCSF and expressed and purified as described in ref. 23. . . .
  24. Burdzy, A., Noyes, K. T., Valinluck, V. & Sowers, L. C. Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA. Nucleic Acids Res. 30, 4068-4074 , .
    • . . . This procedure was adapted from ref. 24. dUMP (225 mg) was dissolved twice in 5 ml of D2O (>99.96 D atom) under Ar gas, and evaporated under vacuum (<50 mtorr) to dryness to reduce proton contamination . . .
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