Recent Insights Into Mechanism and Structure of MEP Pathway Enzymes and Implications for Inhibition Strategies

DeColli, Alicia A.; Johnston, Melanie L.; Meyers, Caren L. Freel

doi:10.1016/b978-0-12-409547-2.14710-9

Cited by 4 publications

(19 citation statements)

References 220 publications

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“…1-Deoxy- d -xylulose 5-phosphate synthase (DXPS) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the formation of DXP from pyruvate and d -glyceraldehyde 3-phosphate ( d -GAP) in bacteria, apicomplexan parasites, and plants. − Due to its importance to bacterial metabolism and absence from human metabolism, DXPS is a promising antibacterial drug target. − Its product, DXP, is a branch-point metabolite that feeds into three essential pathways for metabolite synthesis: ThDP, pyridoxal phosphate, and isoprenoid biosynthesis from isopentenyl diphosphate and dimethylallyl diphosphate (Figure ). , Bacterial pathogen metabolism rapidly changes in response to environmental conditions. − During infection, fluctuating host environments force bacteria to adapt through differential gene expression, metabolic regulation, and modulation of enzyme activity to ensure survival. − Given its role in essential cofactor and isoprenoid biosynthesis, we hypothesize that DXPS is critical for such bacterial adaptations.…”

Section: Introductionmentioning

confidence: 99%

“…The catalytic promiscuity of DXPS is evidenced by its oxygenase activity in which pyruvate is converted to peracetate via the ThDP-enamine radical and superoxide formed by electron transfer from the carbanion to O 2 . We have hypothesized that this gated mechanism could poise DXPS to perform alternative chemistries in response to cellular cues during bacterial adaptation. , …”

Section: Introductionmentioning

confidence: 99%

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Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

et al. 2022

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The bacterial enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate and d-glyceraldehyde 3-phosphate (d-GAP) in a thiamin diphosphate (ThDP)-dependent manner. In addition to its role in isoprenoid biosynthesis, DXP is required for ThDP and pyridoxal phosphate biosynthesis. Due to its function as a branch-point enzyme and its demonstrated substrate and catalytic promiscuity, we hypothesize that DXPS could be key for bacterial adaptation in the dynamic metabolic landscape during infection. Prior work in the Freel Meyers laboratory has illustrated that DXPS displays relaxed specificity toward donor and acceptor substrates and varies acceptor specificity according to the donor used. We have reported that DXPS forms dihydroxyethyl (DHE)ThDP from ketoacid or aldehyde donor substrates via decarboxylation and deprotonation, respectively. Here, we tested other DHE donors and found that DXPS cleaves d-xylulose 5-phosphate (X5P) at C2–C3, producing DHEThDP through a third mechanism involving d-GAP elimination. We interrogated DXPS-catalyzed reactions using X5P as a donor substrate and illustrated (1) production of a semi-stable enzyme-bound intermediate and (2) O2, H+, and d-erythrose 4-phosphate act as acceptor substrates, highlighting a new transketolase-like activity of DXPS. Furthermore, we examined X5P binding to DXPS and suggest that the d-GAP binding pocket plays a crucial role in X5P binding and turnover. Overall, this study reveals a ketose-cleavage reaction catalyzed by DXPS, highlighting the remarkable flexibility for donor substrate usage by DXPS compared to other C–C bond-forming enzymes.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

et al. 2022

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“… 2 , 13 , 14 These structural and mechanistic features of DXPS have guided the development of inhibitors that display selective inhibition of DXPS over related ThDP enzymes. 7 , 8 , 21 − 23 …”

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“…The central metabolite DXP feeds into the biosynthesis of vitamins ThDP and pyridoxal phosphate as well as isoprenoid precursors isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP) (Figure ). , While DXP synthase (DXPS) is essential in many bacterial pathogens, it is absent in humans, highlighting its potential as an antibacterial drug target. − DXPS has unique structural and mechanistic features compared to other ThDP-dependent enzymes − that can be exploited to specifically target DXPS. Among these features are its large active site volume and unique domain arrangement compared to those of the related human ThDP-dependent enzymes transketolase (TK) and the E1 subunit of pyruvate dehydrogenase, ,− as well as a random sequential mechanism involving ternary complex formation. ,,, Distinct from other ThDP-dependent enzymes, the first enzyme-bound intermediate, C2α-lactyl-ThDP (LThDP), is stabilized on DXPS in the absence of the acceptor substrates d -GAP and O 2 . ,, d -GAP plays two roles on DXPS: as a trigger of LThDP decarboxylation upon binding to the E–LThDP complex and as an acceptor substrate in the subsequent carboligation step to produce DXP (Figure ).…”

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confidence: 99%

“…Among these features are its large active site volume and unique domain arrangement compared to those of the related human ThDP-dependent enzymes transketolase (TK) and the E1 subunit of pyruvate dehydrogenase, ,− as well as a random sequential mechanism involving ternary complex formation. ,,, Distinct from other ThDP-dependent enzymes, the first enzyme-bound intermediate, C2α-lactyl-ThDP (LThDP), is stabilized on DXPS in the absence of the acceptor substrates d -GAP and O 2 . ,, d -GAP plays two roles on DXPS: as a trigger of LThDP decarboxylation upon binding to the E–LThDP complex and as an acceptor substrate in the subsequent carboligation step to produce DXP (Figure ). ,, These structural and mechanistic features of DXPS have guided the development of inhibitors that display selective inhibition of DXPS over related ThDP enzymes. ,,− …”

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Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Johnston

Meyers

2021

Biochemistry

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The thiamin diphosphate-dependent enzyme 1-deoxy- d -xylulose 5-phosphate synthase (DXPS) catalyzes the formation of DXP from pyruvate (donor) and d -glyceraldehyde 3-phosphate ( d -GAP, acceptor). DXPS is essential in bacteria but absent in human metabolism, highlighting it as a potential antibacterial drug target. The enzyme possesses unique structural and mechanistic features that enable development of selective inhibition strategies and raise interesting questions about DXPS function in bacterial pathogens. DXPS distinguishes itself within the ThDP enzyme class by its exceptionally large active site and random sequential mechanism in DXP formation. In addition, DXPS displays catalytic promiscuity and relaxed acceptor substrate specificity, yet previous studies have suggested a preference for pyruvate as the donor substrate when d -GAP is the acceptor substrate. However, such donor specificity studies are potentially hindered by a lack of knowledge about specific, alternative donor–acceptor pairs. In this study, we exploited the promiscuous oxygenase activity of DXPS to uncover alternative donor substrates for DXPS. Characterization of glycolaldehyde, hydroxypyruvate, and ketobutyrate as donor substrates revealed differences in stabilization of enzyme-bound intermediates and acceptor substrate usage, illustrating the influence of the donor substrate on reaction mechanism and acceptor specificity. In addition, we found that DXPS prevents abortive acetyl-ThDP formation from a DHEThDP carbanion/enamine intermediate, similar to transketolase, supporting the potential physiological relevance of this intermediate on DXPS. Taken together, these results offer clues toward alternative roles for DXPS in bacterial pathogen metabolism.

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Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog

Coco,

Toci,

Chen

et al. 2024

ACS Infect. Dis.

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New antimicrobial strategies are needed to address pathogen resistance to currently used antibiotics. Bacterial central metabolism is a promising target space for the development of agents that selectively target bacterial pathogens. 1-Deoxy-D-xylulose 5-phosphate synthase (DXPS) converts pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) to DXP, which is required for synthesis of essential vitamins and isoprenoids in bacterial pathogens. Thus, DXPS is a promising antimicrobial target. Toward this goal, our lab has demonstrated selective inhibition of Escherichia coli DXPS by alkyl acetylphosphonate (alkylAP)based bisubstrate analogs that exploit the requirement for ternary complex formation in the DXPS mechanism. Here, we present the first DXPS structure with a bisubstrate analog bound in the active site. Insights gained from this cocrystal structure guided structure−activity relationship studies of the bisubstrate scaffold. A low nanomolar inhibitor (compound 8) bearing a gem-dibenzyl glycine moiety conjugated to the acetylphosphonate pyruvate mimic via a triazole-based linker emerged from this study. Compound 8 was found to exhibit slow, tight-binding inhibition, with contacts to E. coli DXPS residues R99 and R478 demonstrated to be important for this behavior. This work has discovered the most potent DXPS inhibitor to date and highlights a new role of R99 that can be exploited in future inhibitor designs toward the development of a novel class of antimicrobial agents.

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Recent Insights Into Mechanism and Structure of MEP Pathway Enzymes and Implications for Inhibition Strategies

Cited by 4 publications

References 220 publications

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

Antibacterial Target DXP Synthase Catalyzes the Cleavage of d-Xylulose 5-Phosphate: a Study of Ketose Phosphate Binding and Ketol Transfer Reaction

Revealing Donor Substrate-Dependent Mechanistic Control on DXPS, an Enzyme in Bacterial Central Metabolism

Potent Inhibition of E. coli DXP Synthase by a gem-Diaryl Bisubstrate Analog

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