Dataset Information

Probing the Structural Dynamics of the Plasmodium falciparum Tunneling-Fold Enzyme 6-Pyruvoyl Tetrahydropterin Synthase to Reveal Allosteric Drug Targeting Sites.

ABSTRACT: The de novo folate synthesis pathway is a well-established drug target in the treatment of many infectious diseases. Antimalarial antifolate drugs have proven to be effective against malaria, however, rapid drug resistance has emerged on the two primary targeted enzymes: dihydrofolate reductase and dihydroptoreate synthase. The need to identify alternative antifolate drugs and novel metabolic targets is of imminent importance. The 6-pyruvol tetrahydropterin synthase (PTPS) enzyme belongs to the tunneling fold protein superfamily which is characterized by a distinct central tunnel/cavity. The enzyme catalyzes the second reaction step of the parasite's de novo folate synthesis pathway and is responsible for the conversion of 7,8-dihydroneopterin to 6-pyruvoyl-tetrahydropterin. In this study, we examine the structural dynamics of Plasmodium falciparum PTPS using the anisotropic network model, to elucidate the collective motions that drive the function of the enzyme and identify potential sites for allosteric modulation of its binding properties. Based on our modal analysis, we identified key sites in the N-terminal domains and central helices which control the accessibility to the active site. Notably, the N-terminal domains were shown to regulate the open-to-closed transition of the tunnel, via a distinctive wringing motion that deformed the core of the protein. We, further, combined the dynamic analysis with motif discovery which revealed highly conserved motifs that are unique to the Plasmodium species and are located in the N-terminal domains and central helices. This provides essential structural information for the efficient design of drugs such as allosteric modulators that would have high specificity and low toxicity as they do not target the PTPS active site that is highly conserved in humans.

SUBMITTER: Khairallah A

PROVIDER: S-EPMC7546909 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Probing the Structural Dynamics of the <i>Plasmodium falciparum</i> Tunneling-Fold Enzyme 6-Pyruvoyl Tetrahydropterin Synthase to Reveal Allosteric Drug Targeting Sites.

Khairallah Afrah A Ross Caroline J CJ Tastan Bishop Özlem Ö

Frontiers in molecular biosciences 20200925

The <i>de novo</i> folate synthesis pathway is a well-established drug target in the treatment of many infectious diseases. Antimalarial antifolate drugs have proven to be effective against malaria, however, rapid drug resistance has emerged on the two primary targeted enzymes: dihydrofolate reductase and dihydroptoreate synthase. The need to identify alternative antifolate drugs and novel metabolic targets is of imminent importance. The 6-pyruvol tetrahydropterin synthase (PTPS) enzyme belongs ...[more]

PMID: 33102524

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Project description:BackgroundPyruvoyl Tetrahydropterin Synthase (PTPS) Deficiency is the most common form of BH4 deficiency resulting in hyperphenylalaninemia. It can have variable clinical severity and there is limited information on the clinical presentation, natural history and effectiveness of newborn screening for this condition.MethodsRetrospective data (growth and clinical parameters, biochemical and genetic testing results, treatment) were collected from 19 patients with PTPS deficiency in different centers, to evaluate biochemical and clinical outcomes. Descriptive statistics was used for qualitative variables, while linear regression analysis was used to correlate quantitative variables.ResultsPatients with PTPS deficiency had an increased incidence of prematurity (4/18) with an average gestational age only mildly reduced (37.8 ± 2.4 weeks) and low birth weight (-1.14 ± 0.97 SD below that predicted for gestational age). With time, weight and height approached normal.ValuesAll patients were identified by newborn screening for an elevated phenylalanine level. However, phenylalanine levels were normal in two whose testing was performed at or before 24 h of age. Sapropterin dihydrochloride treatment normalized phenylalanine levels. Molecular testing identified novel variants in the PTS gene, some of which present in more than one affected family. The neurotransmitter derivatives 5-hydroxyindoleacetic acid (5HIAA) and homovanillic acid (HVA) in the CSF were decreased in most cases except in 2 families with the peripheral form of PTPS deficiency. With time, HVA and 5HIAA became abnormally low in two of these patients requiring therapy. Prolactin (whose secretion is inhibited by dopamine) levels were elevated in several patients with PTPS deficiency and inversely correlated with the z-scores for height (p < 0.01) and weight (p < 0.05). Most patients with PTPS deficiency had delayed development early in life, improving around school age with IQs mostly in the normal range, with a small decline in older individuals. From a neurological standpoint, most patients had normal brain MRI and minor EEG anomalies, although some had persistent neurological symptoms.DiscussionPatients with PTPS deficiency have not only an increased incidence of prematurity, but also decreased birth weight when corrected for gestational age. Hyperphenylalaninemia can be absent in the first day of life. Therapy with sapropterin dihydrochloride normalizes phenylalanine levels and neurotransmitter precursors can improve CSF neurotransmitter metabolites levels. Insufficient dopaminergic stimulation (as seen from elevated prolactin) might result in decreased height in patients with PTPS deficiency. Despite early delays in development, many patients can achieve independence in adult life, with usually normal neuroimaging and EEG.

Project description:Plasmodium falciparum thymidylate synthase-dihydrofolate reductase (TS-DHFR) is an essential enzyme in nucleotide biosynthesis and a validated molecular drug target in malaria. Because P. falciparum TS and DHFR are highly homologous to their human counterparts, existing active-site antifolate drugs can have dose-limiting toxicities. In humans, TS and DHFR are two separate proteins. In P. falciparum, however, TS-DHFR is bifunctional, with both TS and DHFR active sites on a single polypeptide chain of the enzyme. Consequently, P. falciparum TS-DHFR contains unique distant or nonactive regions that might modulate catalysis: (1) an N-terminal tail and (2) a linker region tethering DHFR to TS, and encoding a crossover helix that forms critical electrostatic interactions with the DHFR active site. The role of these nonactive sites in the bifunctional P. falciparum TS-DHFR is unknown. We report the first in-depth, pre-steady-state kinetic characterization of the full-length, wild-type (WT) P. falciparum TS-DHFR enzyme and probe the role of distant, nonactive regions through mutational analysis. We show that the overall rate-limiting step in the WT P. falciparum TS-DHFR enzyme is TS catalysis. We further show that if TS is in an activated (liganded) conformation, the DHFR rate is 2-fold activated, from 60 s-1 to 130 s-1 in the WT enzyme. The TS rate is also reciprocally activated by approximately 1.5-fold if DHFR is in an activated, ligand-bound conformation. Mutations to the linker region affect neither catalytic rate nor domain-domain communication. Deletion of the N-terminal tail, although in a location remote from the active site, decreases the DHFR single rate and the bifunctional TS-DHFR rate by a factor of 2. The 2-fold activation of the DHFR rate by TS ligands remains intact, although even the activated N-terminal mutant has just half the DHFR activity of the WT enzyme. However, the reciprocal communication between TS active site and DHFR ligands is impaired in N-terminal mutants. Surprisingly, deletion of the analogous N-terminal tail in Leishmania major TS-DHFR causes a 3-fold enhancement of the DHFR rate from approximately 14 s-1 to approximately 40 s-1. In summary, our results demonstrate a complex interplay of domain-domain communication and nonactive-site modulation of catalysis in P. falciparum TS-DHFR. Furthermore, each parasitic TS-DHFR is activated by unique mechanisms, modulated by their nonactive site regions. Finally, our studies suggest the N-terminal tail of P. falciparum TS-DHFR is a highly selective, novel target for potential antifolate development in malaria.

Dataset Information

Probing the Structural Dynamics of the Plasmodium falciparum Tunneling-Fold Enzyme 6-Pyruvoyl Tetrahydropterin Synthase to Reveal Allosteric Drug Targeting Sites.

Publications

Probing the Structural Dynamics of the <i>Plasmodium falciparum</i> Tunneling-Fold Enzyme 6-Pyruvoyl Tetrahydropterin Synthase to Reveal Allosteric Drug Targeting Sites.

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