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Revealing substrate promiscuity of 1-deoxy-D-xylulose 5-phosphate synthase.

ABSTRACT: A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate.

SUBMITTER: Brammer LA

PROVIDER: S-EPMC2761658 | biostudies-literature | 2009 Oct

REPOSITORIES: biostudies-literature

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Revealing substrate promiscuity of 1-deoxy-D-xylulose 5-phosphate synthase.

Brammer Leighanne A LA Meyers Caren Freel CF

Organic letters 20091001 20

A study of DXP synthase has revealed flexibility in the acceptor substrate binding pocket for nonpolar substrates and has uncovered new details of the catalytic mechanism to show that pyruvate can act as both donor and acceptor substrate. ...[more]

PMID: 19778006

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Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.

Project description:The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.

| S-EPMC3675625 | biostudies-literature

Mechanistic Studies of 1-Deoxy-D-Xylulose-5-Phosphate Synthase from Deinococcus radiodurans.

Project description:The non-mevalonate dependent (NMVA) pathway for the biosynthesis of isopentenyl pyrophosphate and dimethylallyl pyrophosphate is the sole source of these terpenoids for the production of isoprenoids in the apicomplexan parasites, in many eubacteria, and in plants. The absence of this pathway in higher organisms has opened a new platform for the development of novel antibiotics and anti-malarials. The enzyme catalyzing the first step of the NMVA pathway is 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). DXPS catalyzes the thiamine pyrophosphate- and Mg (II)-dependent conjugation of pyruvate and D-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose-5-phosphate and CO2. The kinetic mechanism of DXPS from Deinococcus radiodurans most consistent with our data is random sequential as shown using a combination of kinetic analysis and product and dead-end inhibition studies. The role of active site amino acids, identified by sequence alignment to other DXPS proteins, was probed by constructing and analyzing the catalytic efficacy of a set of targeted site-directed mutants.

| S-EPMC5851014 | biostudies-literature

1-Deoxy-D-xylulose 5-phosphate synthase catalyzes a novel random sequential mechanism.

Project description:Emerging resistance of human pathogens to anti-infective agents make it necessary to develop new agents to treat infection. The methylerythritol phosphate pathway has been identified as an anti-infective target, as this essential isoprenoid biosynthetic pathway is widespread in human pathogens but absent in humans. The first enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase, catalyzes the formation of DXP via condensation of D-glyceraldehyde 3-phosphate (D-GAP) and pyruvate in a thiamine diphosphate-dependent manner. Structural analysis has revealed a unique domain arrangement suggesting opportunities for the selective targeting of DXP synthase; however, reports on the kinetic mechanism are conflicting. Here, we present the results of tryptophan fluorescence binding and kinetic analyses of DXP synthase and propose a new model for substrate binding and mechanism. Our results are consistent with a random sequential kinetic mechanism, which is unprecedented in this enzyme class.

| S-EPMC3196101 | biostudies-literature

Targeting the Unique Mechanism of Bacterial 1-Deoxy-d-xylulose-5-phosphate Synthase.

Project description:The bacterial metabolite 1-deoxy-d-xyulose 5-phosphate (DXP) is essential in bacterial central metabolism feeding into isoprenoid, thiamin diphosphate (ThDP), and pyridoxal phosphate de novo biosynthesis. Halting its production through the inhibition of DXP synthase is an attractive strategy for the development of novel antibiotics. Recent work has revealed that DXP synthase utilizes a unique random sequential mechanism that requires formation of a ternary complex among pyruvate-derived C2α-lactylthiamin diphosphate (LThDP), d-glyceraldehyde 3-phosphate (d-GAP), and enzyme, setting it apart from all other known ThDP-dependent enzymes. Herein, we describe the development of bisubstrate inhibitors bearing an acetylphosphonate (AP) pyruvate mimic and a distal negative charge mimicking the phosphoryl group of d-GAP, designed to target the unique form of DXP synthase that binds LThDP and d-GAP in a ternary complex. A d-phenylalanine-derived triazole acetylphosphonate (d-PheTrAP) emerged as the most potent inhibitor in this series, displaying slow, tight-binding inhibition with a Ki* of 90 ± 10 nM, forward ( k1) and reverse ( k2) isomerization rates of 1.1 and 0.14 min-1, respectively, and exquisite selectivity (>15000-fold) for DXP synthase over mammalian pyruvate dehydrogenase. d-PheTrAP is the most potent, selective DXP synthase inhibitor described to date and represents the first inhibitor class designed specifically to exploit the unique E-LThDP-GAP ternary complex in ThDP enzymology.

| S-EPMC6057799 | biostudies-literature

Selective inhibition of E. coli 1-deoxy-D-xylulose-5-phosphate synthase by acetylphosphonates().

Project description:DXP synthase catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate, an essential precursor in pathogen isoprenoid biosynthesis. The selective inhibition of this ThDP-dependent transformation is a challenging goal in the development of isoprenoid biosynthesis inhibitors. Potent, selective inhibitors could lead to new anti-infective agents. Here, we demonstrate selective inhibition of E. coli DXP synthase by butylacetylphosphonate.

| S-EPMC3544079 | biostudies-literature

Phage Display on the Anti-infective Target 1-Deoxy-d-xylulose-5-phosphate Synthase Leads to an Acceptor-Substrate Competitive Peptidic Inhibitor.

Project description:Enzymes of the 2-C-methyl-d-erythritol-4-phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1-deoxy-d-xylulose-5-phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false-positive hits. The enriched peptide binder P12 emerged as a substrate (d-glyceraldehyde-3-phosphate)-competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor- and acceptor-substrate-binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.

| S-EPMC5814854 | biostudies-literature

Structure-activity relationships of compounds targeting mycobacterium tuberculosis 1-deoxy-D-xylulose 5-phosphate synthase.

Project description:We report on a target-based approach to identify possible Mycobacterium tuberculosis DXS inhibitors from the structure of a known transketolase inhibitor. A small focused library of analogs was assembled in order to begin elucidating some meaningful structure-activity relationships of 3-(4-chloro-phenyl)-5-benzyl-4H-pyrazolo[1,5-a]pyrimidin-7-one. Ultimately we found that 2-methyl-3-(4-fluorophenyl)-5-(4-methoxy-phenyl)-4H-pyrazolo[1,5-a]pyrimidin-7-one, although still weak, was able to inhibit M. tuberculosis DXS with an IC(50) of 10.6 microM.

| S-EPMC4784473 | biostudies-literature

Functional Characterization of the 1-Deoxy-D-Xylulose 5-Phosphate Synthase Genes in Morus notabilis.

Project description:Terpenoids are considered to be the largest group of secondary metabolites and natural products. Studies have revealed 1-deoxy-D-xylulose 5-phosphate synthase (DXS) is the first and rate-limiting enzyme in the plastidial methylerythritol phosphate pathway, which produces isopentenyl diphosphate and its isoform dimethylallyl diphosphate as terpenoid biosynthesis precursors. Mulberry (Morus L.) is an economically and ecologically important perennial tree with diverse secondary metabolites, including terpenoids that protect plants against bacteria and insects and may be useful for treating human diseases. However, there has been relatively little research regarding DXS genes in mulberry and other woody plant species. In this study, we cloned and functionally characterized three Morus notabilis DXS genes (MnDXS1, MnDXS2A, and MnDXS2B). Bioinformatics analyses indicated MnDXS1 belongs to clade 1, whereas MnDXS2A and MnDXS2B are in clade 2. The three encoded MnDXS proteins are localized to chloroplasts. Additionally, substantial differences in MnDXS expression patterns were observed in diverse tissues and in response to insect feeding and methyl jasmonate treatment. Moreover, overexpression of MnDXS1 in Arabidopsis thaliana increased the gibberellic acid content and resulted in early flowering, whereas overexpression of MnDXS2A enhanced root growth and increased the chlorophyll and carotenoid content. Our findings indicate that MnDXS functions vary among the clades, which may be useful for further elucidation of the functions of the DXS genes in mulberry.

| S-EPMC7396507 | biostudies-literature

Hydroxybenzaldoximes Are D-GAP-Competitive Inhibitors of E. coli 1-Deoxy-D-Xylulose-5-Phosphate Synthase.

Project description:1-Deoxy-D-xylulose 5-phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low-micromolar inhibitors, competitive against D-glyceraldehyde 3-phosphate (D-GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D-GAP-competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.

| S-EPMC4609000 | biostudies-literature

Characterization of a functionally active recombinant 1-deoxy-D-xylulose-5-phosphate synthase from Babesia bovis.

Project description:The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme has been characterized in other species, but not in the genus Babesia, which causes major losses in the livestock industries worldwide. Therefore, we isolated, cloned and expressed the wild-type B. bovis dxs cDNA in Escherichia coli and evaluated its enzymatic activity in vitro. DNA sequence analysis revealed an open reading frame of 2061 bp capable of encoding a polypeptide of 686 amino acid residues with a calculated isoelectric point of pH 6.93 and a molecular mass of 75 kDa. The expressed soluble recombinant fusion DXS protein was approximately 78 kDa, which is similar to the native enzyme identified from the parasite merozoite using anti-rDXS serum. The recombinant fusion DXS enzyme exhibited Km values of 380 ± 46 µM and 790 ± 52 µM for D,L-glyceraldehyde 3-phosphate and pyruvate, respectively. In this work, we present the first cloning, expression and characterization of DXS enzyme from B. bovis.

| S-EPMC4143643 | biostudies-literature

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