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

Abstract

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.

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Figure 1: Thymidylate synthase mechanisms.
Figure 2: Crystal structures of the FDTS–FAD–dUMP complex.
Figure 3: 2H NMR (a, c) and 1H NMR (b, d) spectra of dTMP produced in the FDTS catalysed reaction of dUMP in D2O (experiment A in Fig. 4 ).
Figure 4: Hydride flow.

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Atomic coordinates and structure factor files have been deposited with the Protein Data Bank under the accession codes 3g4a and 3g4c.

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Acknowledgements

This work was supported by NIH R01 GM065368 and NSF CHE 0715448 to A.K., the Iowa Center for Biocatalysis and Bioprocessing to E.M.K., NIH R01 GM61087 to B.A.P., NIH training grant GM08270 to J.A.C., and JCSG grant U54GM074898 to S.A.L. Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL), a national user facility operated by Stanford University on behalf of DOE, OBER. The SSRL Structural Molecular Biology Program is supported by DOE, OBER and by NIH, NCRR, Biomedical Technology Program and NIGMS.

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Correspondence to Amnon Kohen.

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This file contains Supplementary Tables S1-S2, a Supplementary Discussion, Supplementary Notes and Data for Supplementary Figures S1-S3 with Legends and Supplementary References. (PDF 237 kb)

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Koehn, E., Fleischmann, T., Conrad, J. et al. An unusual mechanism of thymidylate biosynthesis in organisms containing the thyX gene. Nature 458, 919–923 (2009). https://doi.org/10.1038/nature07973

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