Dataset Information

DUMP/F-dUMP Binding to Thymidylate Synthase: Human Versus Mycobacterium tuberculosis.

ABSTRACT: Thymidylate synthase is an enzyme that catalyzes deoxythymidine monophosphate (dTMP) synthesis from substrate deoxyuridine monophosphate (dUMP). Thymidylate synthase of Mycobacterium tuberculosis (MtbThyX) is structurally distinct from its human analogue human thymidylate synthase (hThyA), thus drawing attention as an attractive drug target for combating tuberculosis. Fluorodeoxyuridylate (F-dUMP) is a successful inhibitor of both MtbThyX and hThyA, thus limited by poor selectivity. Understanding the dynamics and energetics associated with substrate/inhibitor binding to thymidylate synthase in atomic details remains a fundamental unsolved problem, which is necessary for a new selective inhibitor design. Structural studies of MtbThyX and hThyA bound substrate/inhibitor complexes not only revealed the extensive specific interaction network between protein and ligands but also opened up the possibility of directly computing the energetics of the substrate versus inhibitor recognition. Using experimentally determined structures as a template, we report extensive computer simulations (?4.5 ?s) that allow us to quantitatively estimate ligand selectivity (dUMP vs F-dUMP) by MtbThyX and hThyA. We show that MtbThyX prefers deprotonated dUMP (enolate form) as the substrate, whereas hThyA binds to the keto form of dUMP. Computed energetics clearly show that MtbThyX is less selective between dUMP and F-dUMP, favoring the latter, relative to hThyA. The simulations reveal the role of tyrosine at position 135 (Y135) of hThyA in amplifying the selectivity. The protonation state of the pyrimidine base of the ligand (i.e., keto or enolate) seems to have no role in MtbThyX ligand selectivity. A molecular gate (consists of Y108, K165, H203, and a water molecule) restricts water accessibility and offers a desolvated dry ligand-binding pocket for MtbThyX. The ligand-binding pocket of hThyA is relatively wet and exposed to bulk water.

SUBMITTER: Gaurav K

PROVIDER: S-EPMC7376888 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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dUMP/F-dUMP Binding to Thymidylate Synthase: Human Versus Mycobacterium tuberculosis.

Gaurav Kumar K Adhikary Tiasha T Satpati Priyadarshi P

ACS omega 20200706 28

Thymidylate synthase is an enzyme that catalyzes deoxythymidine monophosphate (dTMP) synthesis from substrate deoxyuridine monophosphate (dUMP). Thymidylate synthase of Mycobacterium tuberculosis (MtbThyX) is structurally distinct from its human analogue human thymidylate synthase (hThyA), thus drawing attention as an attractive drug target for combating tuberculosis. Fluorodeoxyuridylate (F-dUMP) is a successful inhibitor of both MtbThyX and hThyA, thus limited by poor selectivity ...[more]

PMID: 32715203

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Project description:Mycobacterium tuberculosis kills approximately 2 million people each year and presents an urgent need to identify new targets and new antitubercular drugs. Thymidylate synthase (TS) enzymes from other species offer good targets for drug development and the M. tuberculosis genome contains two putative TS enzymes, a conventional ThyA and a flavin-based ThyX. In M. tuberculosis, both TS enzymes have been implicated as essential for growth, either based on drug-resistance studies or genome-wide mutagenesis screens. To facilitate future small molecule inhibitors against these proteins, a detailed enzymatic characterization was necessary.After cloning, overexpression, and purification, the thymidylate-synthesizing ability of ThyA and ThyX gene products were directly confirmed by HPLC analysis of reaction products and substrate saturation kinetics were established. 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was a potent inhibitor of both ThyA and ThyX, offering important clues to double-targeting strategies. In contrast, the folate-based 1843U89 was a potent inhibitor of ThyA but not ThyX suggesting that it should be possible to find ThyX-specific antifolates. A turnover-dependent kinetic assay, combined with the active-site titration approach of Ackermann and Potter, revealed that both M. tuberculosis enzymes had very low k(cat) values. One possible explanation for the low catalytic activity of M. tuberculosis ThyX is that its true biological substrates remain to be identified. Alternatively, this slow-growing pathogen, with low demands for TMP, may have evolved to down-regulate TS activities by altering the turnover rate of individual enzyme molecules, perhaps to preserve total protein quantities for other purposes. In many organisms, TS is often used as a part of larger complexes of macromolecules that control replication and DNA repair.Thus, the present enzymatic characterization of ThyA and ThyX from M. tuberculosis provides a framework for future development of cell-active inhibitors and the biological roles of these TS enzymes in M. tuberculosis.

Dataset Information

DUMP/F-dUMP Binding to Thymidylate Synthase: Human Versus Mycobacterium tuberculosis.

Publications

dUMP/F-dUMP Binding to Thymidylate Synthase: Human Versus <i>Mycobacterium tuberculosis</i>.

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