Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR

Anderson, Karen S.

doi:10.1093/protein/gzx004

Cited by 16 publications

(10 citation statements)

References 46 publications

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“…The two active sites are about 40 Å apart, separated by a solvent‐exposed tunnel. This tunnel is lined by an excess of positively charged residues , suggesting that substrate channeling of the negatively charged acyl‐CoA molecules via electrostatic interactions with the positively charged tunnel surface could be important, as described for the thymidylate synthase–dihydrofolate reductase bifunctional enzyme .…”

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confidence: 91%

Structural enzymology comparisons of multifunctional enzyme, type‐1 (MFE1): the flexibility of its dehydrogenase part

et al. 2017

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Multifunctional enzyme, type‐1 ( MFE 1) is a monomeric enzyme with a 2E‐enoyl‐CoA hydratase and a 3S‐hydroxyacyl‐CoA dehydrogenase ( HAD ) active site. Enzyme kinetic data of rat peroxisomal MFE 1 show that the catalytic efficiencies for converting the short‐chain substrate 2E‐butenoyl‐CoA into acetoacetyl‐CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl‐CoA (to the hydratase active site) and the very similar mode of binding of NAD + and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE 1 are correlated. The possible importance of the conformational flexibility of MFE 1 for its biocatalytic properties is discussed. Database Structural data are available in PDB database under the accession number 5MGB .

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confidence: 91%

Structural enzymology comparisons of multifunctional enzyme, type‐1 (MFE1): the flexibility of its dehydrogenase part

et al. 2017

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“…Thymidylate synthase (TS, UniProt entry P0A884) is a dimeric enzyme responsible for the synthesis of the sole de novo source of deoxythymidine monophosphate (dTMP) in most organisms. Some pathogenic microbes utilize two forms of TS, one canonical thy A type and the other a flavin-dependent enzyme, or thy X, type. , In addition, although some organisms use so-called bifunctional enzymes, where TS and another enzyme in the folate metabolism cycle, dihydrofolate reductase, are encoded on the same polypeptide, the fold and primary sequences of thy A-type TS enzymes are highly conserved in all forms studied to date. TS uses a multiple-step reaction coordinate along which both a methylene and hydride are donated from the methylene tetrahydrofolate (mTHF) cofactor to the deoxyuridine monophosphate (dUMP) substrate.…”

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confidence: 99%

Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

et al. 2019

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Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for, nor the mechanism of this phenomenon is understood. We used a combined NMR and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate limiting, and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apo enzyme (lig 0), the saturated holo-enzyme (lig 2), and the typically elusive singly bound (lig 1) states. ~40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes greater than 30 Å away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface βsheet, which also represents the shortest path between the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having inter-subunit cross-correlated motions. Simulations identify five hotspot residues, three of which lie at the kink in the unique β-bulge abutting the active sites on either end of the sheet. Inter-strand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig 0 to lig 1 , and ultimately lig 2. Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing symmetrical.

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“…Active sites of DHFR and TS are located on different faces of the TgDHFR-TS structure like other apicomplexan DHFR-TS enzymes. The JR between DHFR and TS moieties in apicomplexans is rather long (55–91 amino acids), whereas it is much shorter or absent in other kinetoplastids such as Trypanosoma brucei, Trypanosoma cruzi, and Leishmania major . It is reported that the JR is essential for the catalytic activity of Pf-, Ch-, and Tg-DHFR-TS enzymes. ,, A crossover helix in JR is found in all apicomplexan enzymes with various lengths.…”

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confidence: 99%

Structural Insight into Effective Inhibitors’ Binding to Toxoplasma gondii Dihydrofolate Reductase Thymidylate Synthase

et al. 2022

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Pyrimethamine (Pyr), a known dihydrofolate reductase (DHFR) inhibitor, has long been used to treat toxoplasmosis caused by Toxoplasma gondii (Tg) infection. However, Pyr is effective only at high doses with associated toxicity to patients, calling for safer alternative treatments. In this study, we investigated a series of Pyr analogues, previously developed as DHFR inhibitors of Plasmodium falciparum bifunctional DHFR-thymidylate synthase (PfDHFR-TS), for their activity against T. gondii DHFR-TS (TgDHFR-TS). Of these, a set of compounds with a substitution at the C 6 position of the pyrimidine ring exhibited high binding affinities (in a low nanomolar range) against TgDHFR-TS and in vitro T. gondii inhibitory activity. Three-dimensional structures of TgDHFR-TS reported here include the ternary complexes with Pyr, P39, or P40. A comparison of these structures showed the minor steric strain between the p-chlorophenyl group of Pyr and Thr83 of TgDHFR-TS. Such a conflict was relieved in the complexes with the two analogues, P39 and P40, explaining their highest binding affinities described herein. Moreover, these structures suggested that the hydrophobic environment in the active-site pocket could be used for drug design to increase the potency and selectivity of antifolate inhibitors. These findings would accelerate the development of new antifolate drugs to treat toxoplasmosis.

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Understanding the molecular mechanism of substrate channeling and domain communication in protozoal bifunctional TS-DHFR

Cited by 16 publications

References 46 publications

Structural enzymology comparisons of multifunctional enzyme, type‐1 (MFE1): the flexibility of its dehydrogenase part

Structural enzymology comparisons of multifunctional enzyme, type‐1 (MFE1): the flexibility of its dehydrogenase part

Inter-Active Site Communication Mediated by the Dimer Interface β-Sheet in the Half-the-Sites Enzyme, Thymidylate Synthase

Structural Insight into Effective Inhibitors’ Binding to Toxoplasma gondii Dihydrofolate Reductase Thymidylate Synthase

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