Mechanistic Characterization of Toxoplasma gondiiThymidylate Synthase (TS-DHFR)-Dihydrofolate Reductase

Johnson, Eric F.; Hinz, Wolfgang; Atreya, Chloé E.; Maley, Frank; Anderson, Karen S.

doi:10.1074/jbc.m206523200

Cited by 38 publications

(53 citation statements)

References 27 publications

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“…In the case of the wild-type enzyme and each of the charge reversal or charge neutralization mutants tested, we observed a burst in consumption of the cofactor, CH 2 H 4 folate at TS (26). Because chemistry is overall ratelimiting at TS (no burst in dUMP consumption or dTMP formation), the observation of a burst in CH 2 H 4 folate consumption signifies the presence of a TS intermediate, most likely the iminium form of CH 2 H 4 folate.…”

Section: Discussionmentioning

confidence: 95%

“…It is thought that this is followed by the formation of an iminium ion involving the bridge methylene and N4 of CH 2 H 4 folate (25). We showed previously, using the wild-type enzyme, that there is a burst in consumption of the cofactor, CH 2 H 4 folate, at TS (26). Because it is known that chemistry is rate-limiting at TS, the observation of a burst in CH 2 H 4 folate consumption signals the accumulation of a TS intermediate, likely the iminium form of CH 2 H 4 folate (22)(23)(24).…”

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confidence: 99%

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Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.Electrostatic channeling is a mechanism proposed based on the crystal structure of bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) 1 from Leishmania major that would enable negatively charged dihydrofolate produced at the TS active site to be handed off along a series of solventexposed lysine and arginine residues to the DHFR active site, where it is converted to tetrahydrofolate (H 4 folate) (1).TS and DHFR are crucial enzymes, found in all species. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2Ј-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate H 4 folate, used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis. Only in protozoal parasites and some plants however, are TS and DHFR found on the same polypeptide chain, leading to the hypothesis that in these organisms, H 2 folate may be channeled from the TS active site to that of DHFR, never equilibrating with bulk solution (Fig. 1A). When the crystal structure of L. major TS-DHFR was solved (2.9 Å resolution), a 40 Å "electrostatic highway" across the surface of the protein was proposed as an explanation for how channeling may occur (1, 2). We sought to test the electrostatic channeling hypothesis with two parallel approaches. In this report we present results of mutagenesis of solvent-exposed basic residues comprising the electrostatic highway. In an earlier paper, we reported the findings from targeted molecular docking searches to identify small molecule inhibitors that bind in this region, as a means to block the putative channel (3).There are precedents for channeling among bifunction...

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Section: Discussionmentioning

confidence: 95%

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confidence: 99%

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Atreya

Johnson

Williamson

et al. 2003

Journal of Biological Chemistry

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“…Although dUMP was recently shown to bind with minimal cooperativity for the E. coli enzyme at 25°C, there are signs of unequal thermodynamics between the two protomers at lower temperatures (16), and indeed data herein show clear intersubunit communication. Moreover, other TS enzymes, and in particular human, seem to show more dramatic cooperativity, suggesting that intersubunit communication is an intrinsic feature of TS (13,(17)(18)(19)(20)(21). To overcome the difficulties of studying symmetric proteins by NMR, we generated a pair of mixed labeled dimers of TS that each have a single functional active site and a single protomer labeled for NMR studies.…”

Section: Significancementioning

confidence: 99%

“…TS catalyzes the synthesis of the sole source of 2′-deoxythymidine-5′-monophosphate (dTMP) via a multistep mechanism involving reductive methylation of dUMP using N5,N10-methylene-5,6,7,8-tetrahydrofolate (mTHF) as both a methylene and hydride donor. In addition, TS is half-the-sites reactive (13)(14)(15), with substrate binding sites separated by 35 Å, leading to an expectation for…”

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Chemical shift imprint of intersubunit communication in a symmetric homodimer

Falk

Sapienza

Lee

2016

Proc. Natl. Acad. Sci. U.S.A.

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Allosteric communication is critical for protein function and cellular homeostasis, and it can be exploited as a strategy for drug design. However, unlike many protein-ligand interactions, the structural basis for the long-range communication that underlies allostery is not well understood. This lack of understanding is most evident in the case of classical allostery, in which a binding event in one protomer is sensed by a second symmetric protomer. A primary reason why study of interdomain signaling is challenging in oligomeric proteins is the difficulty in characterizing intermediate, singly bound species. Here, we use an NMR approach to isolate and characterize a singly ligated state ("lig 1 ") of a homodimeric enzyme that is otherwise obscured by rapid exchange with apo and saturated forms. Mixed labeled dimers were prepared that simultaneously permit full population of the lig 1 state and isotopic labeling of either protomer. Direct visualization of peaks from lig 1 yielded site-specific ligand-state multiplets that provide a convenient format for assessing mechanisms of intersubunit communication from a variety of NMR measurements. We demonstrate this approach on thymidylate synthase from Escherichia coli, a homodimeric enzyme known to be half-the-sites reactive. Resolving the dUMP 1 state shows that active site communication occurs not upon the first dUMP binding, but upon the second. Surprisingly, for many sites, dUMP 1 peaks are found beyond the limits set by apo and dUMP 2 peaks, indicating that binding the first dUMP pushes the enzyme ensemble to further conformational extremes than the apo or saturated forms. The approach used here should be generally applicable to homodimers.A llosteric regulation in proteins is a ubiquitous mechanism for controlling cellular behavior and an attractive strategy for therapeutic development. Even though broadly recognized, longrange communication is not well understood mechanistically (1-4). Although there have been numerous strategies to reveal the structural and dynamic underpinnings of allostery, oddly these strategies have largely focused on complex oligomeric or, alternatively, on small monomeric allosteric proteins. A likely more straightforward approach is to study allosteric mechanisms using simple symmetric homodimeric proteins, which would allow for answering the basic and general question of how the occurrence of an event in one subunit is communicated to another subunit, as occurs in classical multisubunit allosteric proteins. Given the large number of homodimeric proteins involved in cellular regulation (such as growth factors, cytokines, kinases, G protein-coupled receptors, transcription factors, and metabolic proteins), insights into intersubunit communication should be widely beneficial (5).As a key step toward a broad understanding of allosteric mechanisms, it will be important to observe how binding a ligand in one subunit is communicated to a second subunit, even in the absence of conformational change. Although this communication is straightforwar...

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“…Indeed, there is evidence for substrate channeling with both L. major and T. gondii TS-DHFR. In the case of these bifunctional enzymes, unlike with the combination of monofunctional TS ϩ DHFR, H 2 folate produced at the TS active site does not substantially accumulate in solution and there is no lag in product formation at DHFR, suggesting that the TS product does not leave the protein and enter bulk solution before binding at DHFR (5,7,8). Steady-state spectroscopic analysis, used to detect the presence or absence of a lag in NADP ϩ production via the DHFR-catalyzed reduction of H 2 folate formed at TS, also supports substrate channeling by both L. major and T. gondii TS-DHFR (9,10).…”

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confidence: 99%

Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

Atreya¹,

Anderson

2004

Journal of Biological Chemistry

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This study presents a kinetic characterization of the recently crystallized bifunctional thymidylate synthasedihydrofolate reductase (TS-DHFR) enzyme from the apicomplexa parasite, Cryptosporidium hominis. Our study focuses on determination of the C. hominis TS-DHFR kinetic mechanism, substrate channeling behavior, and domain-domain communication. Unexpectedly, the unique mechanistic features of C. hominis TS-DHFR involve the highly conserved TS domain. At 45 s ؊1 , C. hominis TS activity is 10 -40-fold faster than other TS enzymes studied and a new kinetic mechanism was required to simulate C. hominis TS behavior. A large accumulation of dihydrofolate produced at TS and a lag in product formation at DHFR were observed. These observations make C. hominis TS-DHFR the first bifunctional TS-DHFR enzyme studied for which there is clear evidence against dihydrofolate substrate channeling. Furthermore, whereas with Leishmania major TS-DHFR there are multiple lines of evidence for domain-domain communication (ligand binding at one active site affecting activity of the other enzyme), no such effects were observed with C. hominis TS-DHFR.Protozoal parasites such as Cryptosporidium hominis, Plasmodium falciparum, Toxoplasma gondii, and Leishmania major are unusual in that their thymidylate synthase (TS) 1 and dihydrofolate reductase (DHFR) enzymes exist on the same polypeptide as part of a single bifunctional TS-DHFR enzyme.2 TS and DHFR are critical enzymes and established drug targets. TS represents the only means of de novo synthesis of 2Ј-deoxythymidylate (dTMP) for DNA synthesis, via reductive methylation of 2-deoxyuridate (dUMP) with methylene tetrahydrofolate (CH 2 H 4 folate), producing dihydrofolate (H 2 folate) in the process. DHFR catalyzes the reduction of H 2 folate by NADPH to generate tetrahydrofolate (H 4 folate), used for one carbon unit transfer reactions in several biochemical processes, including thymidylate, purine, and amino acid biosynthesis.Almost a decade of research on bifunctional TS-DHFR from protozoal parasites has relied on the only available TS-DHFR crystal structure, that from the kinetoplastid protozoa L. major (1). The recent solution of the crystal structures of TS-DHFR enzymes from two clinically relevant apicomplexan protozoa, P. falciparum and C. hominis, however, has revealed unanticipated deviations from the kinetoplastid model (2, 3). An obvious next question is whether these significant structural differences translate as significant mechanistic differences between parasite classes. We sought to address this question through a detailed kinetic characterization of TS-DHFR from C. hominis, again yielding surprising results.A major conclusion of the recent solution of the apicomplexan TS-DHFR structures is that there exist two families of bifunctional TS-DHFR structures: a short linker family with an Nterminal tail, as in the kinetoplastids (a class that includes Leishmania and the trypanosomes); and a long linker family with a donated or crossover helix, as in the apicomplexan paras...

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Mechanistic Characterization of Toxoplasma gondiiThymidylate Synthase (TS-DHFR)-Dihydrofolate Reductase

Cited by 38 publications

References 27 publications

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Probing Electrostatic Channeling in Protozoal Bifunctional Thymidylate Synthase-Dihydrofolate Reductase Using Site-directed Mutagenesis

Chemical shift imprint of intersubunit communication in a symmetric homodimer

Kinetic Characterization of Bifunctional Thymidylate Synthase-Dihydrofolate Reductase (TS-DHFR) from Cryptosporidium hominis

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