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Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX.

Hunter JH, Gujjar R, Pang CK, Rathod PK - PLoS ONE (2008)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

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Related in: MedlinePlus

RNA binding by M. tuberculosis ThyA and ThyX.M. tuberculosis thymidylate synthases do not bind to their cognate mRNA. 0.35 nM of 32P labeled M. tuberculosis ThyX mRNA (lanes 1 and 2), M. tuberculosis ThyA mRNA (lanes 3 and 4), or P. falciparum DHFR-TS mRNA (lanes 5 and 6) was incubated in the presence or absence (lanes 1, 3, and 5) of purified M. tuberculosis ThyX (3.5 µM, lane 2), M. tuberculosis ThyA (3.5 µM, lane 4), or P. falciparum DHFR-TS (500 nM, lane 6). Unbound RNA was digested with 3 units of RNase T1 prior to electrophoresis [22].
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pone-0002237-g003: RNA binding by M. tuberculosis ThyA and ThyX.M. tuberculosis thymidylate synthases do not bind to their cognate mRNA. 0.35 nM of 32P labeled M. tuberculosis ThyX mRNA (lanes 1 and 2), M. tuberculosis ThyA mRNA (lanes 3 and 4), or P. falciparum DHFR-TS mRNA (lanes 5 and 6) was incubated in the presence or absence (lanes 1, 3, and 5) of purified M. tuberculosis ThyX (3.5 µM, lane 2), M. tuberculosis ThyA (3.5 µM, lane 4), or P. falciparum DHFR-TS (500 nM, lane 6). Unbound RNA was digested with 3 units of RNase T1 prior to electrophoresis [22].

Mentions: Autologous RNA binding by the classical thymidylate synthase, ThyA, can play an important role in pharmacology. Whether a protein binds its own RNA, and whether the resulting autologous translational inhibition can be reversed with enzyme inhibitors, can be important contributing variables to species-specific drug action [21], [22]. Human and P. falciparum ThyA have been shown to bind their cognate mRNA coding sequence and to inhibit their own translation [21]–[23]. The mRNA binding abilities of M. tuberculosis ThyA and ThyX were probed using a gel-shift assay (Fig. 3). Neither ThyA nor ThyX were found to bind their own mRNA. These initial results suggest that the expression levels of M. tuberculosis ThyA and ThyX are not regulated by classic autologous translational feedback loops and instead may be dominated by regulation of RNA transcription.


Kinetics and ligand-binding preferences of Mycobacterium tuberculosis thymidylate synthases, ThyA and ThyX.

Hunter JH, Gujjar R, Pang CK, Rathod PK - PLoS ONE (2008)

RNA binding by M. tuberculosis ThyA and ThyX.M. tuberculosis thymidylate synthases do not bind to their cognate mRNA. 0.35 nM of 32P labeled M. tuberculosis ThyX mRNA (lanes 1 and 2), M. tuberculosis ThyA mRNA (lanes 3 and 4), or P. falciparum DHFR-TS mRNA (lanes 5 and 6) was incubated in the presence or absence (lanes 1, 3, and 5) of purified M. tuberculosis ThyX (3.5 µM, lane 2), M. tuberculosis ThyA (3.5 µM, lane 4), or P. falciparum DHFR-TS (500 nM, lane 6). Unbound RNA was digested with 3 units of RNase T1 prior to electrophoresis [22].
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2386288&req=5

pone-0002237-g003: RNA binding by M. tuberculosis ThyA and ThyX.M. tuberculosis thymidylate synthases do not bind to their cognate mRNA. 0.35 nM of 32P labeled M. tuberculosis ThyX mRNA (lanes 1 and 2), M. tuberculosis ThyA mRNA (lanes 3 and 4), or P. falciparum DHFR-TS mRNA (lanes 5 and 6) was incubated in the presence or absence (lanes 1, 3, and 5) of purified M. tuberculosis ThyX (3.5 µM, lane 2), M. tuberculosis ThyA (3.5 µM, lane 4), or P. falciparum DHFR-TS (500 nM, lane 6). Unbound RNA was digested with 3 units of RNase T1 prior to electrophoresis [22].
Mentions: Autologous RNA binding by the classical thymidylate synthase, ThyA, can play an important role in pharmacology. Whether a protein binds its own RNA, and whether the resulting autologous translational inhibition can be reversed with enzyme inhibitors, can be important contributing variables to species-specific drug action [21], [22]. Human and P. falciparum ThyA have been shown to bind their cognate mRNA coding sequence and to inhibit their own translation [21]–[23]. The mRNA binding abilities of M. tuberculosis ThyA and ThyX were probed using a gel-shift assay (Fig. 3). Neither ThyA nor ThyX were found to bind their own mRNA. These initial results suggest that the expression levels of M. tuberculosis ThyA and ThyX are not regulated by classic autologous translational feedback loops and instead may be dominated by regulation of RNA transcription.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemistry, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: 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.

Methodology/principal findings: 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.

Conclusions/significance: 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.

Show MeSH
Related in: MedlinePlus