Production of recombinant 1‐deoxy‐<scp>d</scp>‐xylulose‐5‐phosphate synthase from <i>Plasmodium vivax</i> in <i>Escherichia coli</i>

Handa, Sumit; Ramamoorthy, Divya; Spradling, Tyler J.; Guida, Wayne C.; Adams, John; Bendinskas, Kestutis; Merkler, David J.

doi:10.1016/j.fob.2013.01.007

Cited by 15 publications

(21 citation statements)

References 35 publications

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“…Overexpression of this metabolic branch point enzyme may have deleterious effects on metabolism. There is evidence that DXP synthase aggregates upon overexpression, 37,38,66 and its overexpression causes toxicity to bacterial cells. 39−41,43,45,67 Despite this, we have successfully shown that basal expression of this enzyme confers resistance to some alkylAPs, albeit to different degrees.…”

Section: Discussionmentioning

confidence: 99%

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

et al. 2017

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1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the thiamin diphosphate (ThDP)-dependent formation of DXP from pyruvate and D-glyceraldehyde 3-phosphate. DXP is at a metabolic branch point in bacteria, feeding into the methylerythritol phosphate pathway to indispensable isoprenoids and acting as a precursor for biosynthesis of essential cofactors in central metabolism, pyridoxal phosphate and ThDP, the latter of which is also required for DXP synthase catalysis. DXP synthase follows a unique random sequential mechanism and possesses an unusually large active site. These features have guided the design of sterically demanding alkylacetylphosphonates (alkylAPs) toward the development of selective DXP synthase inhibitors. alkylAPs studied here display selective, low μM inhibitory activity against DXP synthase. They are weak inhibitors of bacterial growth in standard nutrient rich conditions. However, bacteria are significantly sensitized to most alkylAPs in defined minimal growth medium, with minimal inhibitory concentrations (MICs) ranging from low μM to low mM and influenced by alkyl-chain length. The longest analog (C8) displays the weakest antimicrobial activity and is a substrate for efflux via AcrAB-TolC. The dependence of inhibitor potency on growth environment emphasizes the need for antimicrobial screening conditions that are relevant to the in vivo microbial microenvironment during infection. DXP synthase expression and thiamin supplementation studies offer support for DXP synthase as an intracellular target for some alkylAPs and reveal both the challenges and intriguing aspects of these approaches to study target engagement.

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Section: Discussionmentioning

confidence: 99%

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

et al. 2017

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“…The cloning of Pv DXS was described previously. [30] The cloning of Pf DXR has also been previously described, [31] and this gene was generously donated by Dr. Nobutada Tanaka at Showa University (Japan) for the use in this study.…”

Section: Methodsmentioning

confidence: 99%

“…Cells were harvested by centrifugation, and the cell pellets stored at −80 °C. The expression of Pv DXS [30] and Pf DXR [31] were described previously.…”

Section: Methodsmentioning

confidence: 99%

“…Similar to earlier work on the expression of P. vivax DXS [30] and P. falciparum DXR, [31] the catalytically active forms of P. falciparum DXS and P. vivax DXR have been truncated to their respective catalytic cores, lacking the signaling and transit peptide for each of these enzymes. In addition to recombinant P. falciparum DXS and P. vivax DXR, we also produced the truncated forms of P. vivax DXS and P. falciparum DXR in E. coli by following published protocols.…”

Section: Introductionmentioning

confidence: 99%

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Mechanistic binding insights for 1-deoxy-d-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax

Battistini

Shoji

Handa

et al. 2016

Protein Expression and Purification

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We have successfully truncated and recombinantly-expressed 1-deoxy-D-xylulose-5-phosphate synthase (DXS) from both P. vivax and P. falciparum. We elucidated the order of substrate binding for both of these ThDP-dependent enzymes using steady-state kinetic analyses, dead-end inhibition, and intrinsic tryptophan fluorescence titrations. Both enzymes adhere to a random sequential mechanism with respect to binding of both substrates: pyruvate and D-glyceraldehyde-3-phosphate. These findings are in contrast to other ThDP-dependent enzymes, which exhibit classical ordered and/or ping-pong kinetic mechanisms. A better understanding of the kinetic mechanism for these two Plasmodial enzymes could aid in the development of novel DXS-specific inhibitors that might prove useful in treatment of malaria.

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“…In this study we describe the active site architecture of 1-deoxy-D-xylulose 5-phosphate synthase, a target for antimicrobial agents, herbicides, and antimalarial drugs [22] [23]. A role in catalysis has been assigned to conserved amino acid residues and a model of the catalytic site has been generated which can be of great interest to predict drug-enzyme interactions.…”

Section: Kinetic Studiesmentioning

confidence: 99%

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Querol-Audí¹,

Boronat²,

Centelles³

et al. 2014

JBM

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1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-Derythritol 4-phosphate (MEP)pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.

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Production of recombinant 1‐deoxy‐d‐xylulose‐5‐phosphate synthase from Plasmodium vivax in Escherichia coli

Cited by 15 publications

References 35 publications

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

Challenges and Hallmarks of Establishing Alkylacetylphosphonates as Probes of Bacterial 1-Deoxy-d-xylulose 5-Phosphate Synthase

Mechanistic binding insights for 1-deoxy-d-Xylulose-5-Phosphate synthase, the enzyme catalyzing the first reaction of isoprenoid biosynthesis in the malaria-causing protists, Plasmodium falciparum and Plasmodium vivax

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

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