MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply

Di, Xueni; Ortega-Alarcón, David; Kakumanu, Ramu; Iglésias-Fernández, Javier; Diaz, L. Pedraza; Baidoo, Edward E. K.; Velázquez‐Campoy, Adrián; Rodríguez‐Concepción, Manuel; Pérez-Gil, Jordi

doi:10.1016/j.xplc.2022.100512

Cited by 17 publications

(15 citation statements)

References 46 publications

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“…DXPS is active as a homodimer. A recent study suggests that DXPS activity is regulated via ligand-induced changes to the oligomeric state . To determine the effect of 8 on quaternary structure, we conducted native polyacrylamide gel electrophoresis (PAGE) and analytical gel filtration (aGF) analyses.…”

Section: Resultsmentioning

confidence: 99%

“…Interestingly, analog 8 appears to shift the DXPS oligomer equilibrium, inducing higher-order oligomeric states. Di et al have recently reported ligand-induced monomerization of DXPS by downstream MEP pathway intermediates, 81 suggesting DXPS regulation by reversible reduction of DXPS dimer activity. Although the activity of the higher-order tetramer is unknown, it is conceivable that shifting the oligomer equilibrium could contribute to the inhibitory activity of this enzyme class.…”

Section: ■ Discussionmentioning

confidence: 99%

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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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Section: Resultsmentioning

confidence: 99%

Section: ■ Discussionmentioning

confidence: 99%

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

Coco,

Toci,

Chen

et al. 2024

ACS Infect. Dis.

View full text Add to dashboard Cite

show abstract

“…These efforts relied on augmentation of the endogenous MEP pathway by overexpressing key enzymes; however, titers were limited. Recent studies have demonstrated a conserved, allosteric regulatory mechanism where 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the rate-limiting MEP enzyme, forms inactive aggregates when IPP and DMAPP levels are high (Banerjee and Sharkey 2014; Di et al 2023). The subsequent downregulation of the MEP pathway may be a source of the low titers achieved in the previous C. glutamicum monoterpene production studies.…”

Section: Resultsmentioning

confidence: 99%

“…Lim, Park, and Woo 2020; Li, Xu, and Lu 2021) . Each of these efforts relied upon the overexpression of key components [typically deoxyxylulose 5-phosphate synthase ( dxs ) and isopentenyl diphosphate isomerase ( idi )] within the native MEP pathway, which is subject to endogenous regulation that may limit the isoprenyl precursors, IPP and DMAPP (Banerjee and Sharkey 2014; Li, Xu, and Lu 2021; Kang et al 2014; Di et al 2023). To overcome these potential limitations, (Sasaki et al 2019) leveraged a heterologous MVA-based, episomal bypass pathway to produce isoprenol at titers over 1000 mg/L, providing a promising foundation toward high production for other isoprenoids.…”

Section: Introductionmentioning

confidence: 99%

Development ofCorynebacterium glutamicumas a monoterpene production platform

Luckie,

Kashyap,

Pearson

et al. 2023

Preprint

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Monoterpenes are commonly known for their role in the flavors and fragrances industry and are also gaining attention for other uses like insect repellant and as potential renewable fuels for aviation.Corynebacterium glutamicum,a Generally Recognized as Safe microbe, has been a choice organism in industry for the annual million ton-scale bioproduction of amino acids for more than 50 years; however, efforts to produce monoterpenes inC. glutamicumhave remained relatively limited. In this study, we report a further expansion of theC. glutamicumbiosynthetic repertoire through the development and optimization of a mevalonate-based monoterpene platform. In the course of our plasmid design iterations, we increased flux through the mevalonate-based bypass pathway, measuring isoprenol production as a proxy for monoterpene precursor abundance and demonstrating the highest reported titers inC. glutamicumto date at nearly 1500 mg/L. Our designs also evaluated the effects of backbone, promoter, and GPP synthase homolog origin on monoterpene product titers. Monoterpene production was further improved by disrupting competing pathways for isoprenoid precursor supply and by implementing a biphasic production system to prevent volatilization. With this platform, we achieved 321.1 mg/L of geranoids, 723.6 mg/L of 1,8-cineole, and 227.8 mg/L of linalool. Furthermore, we determined thatC. glutamicumfirst oxidizes geraniol through an aldehyde intermediate before it is asymmetrically reduced to citronellol. Additionally, we demonstrate that the aldehyde reductase, AdhC, possesses additional substrate promiscuity for acyclic monoterpene aldehydes.HighlightsDesign of a mevalonate-based monoterpene production platform inC. glutamicumHighest production titers of geranoids, eucalyptol, and linalool reported inC. glutamicumto dateIdentification of citronellal as an intermediate in the reduction of geraniol to citronellol byC. glutamicum

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“…The change in the rate of DXP turnover, but not in its pool size, likely reflects the role of DXS as the major rate determining step of the MEP pathway 28 , which increases the rate of flux through the pathway when substrate concentrations rise. DXS is also under allosteric 29,30 and proteolytic regulation 31 . The subsequent step, catalyzed by DXP reductoisomerase (DXR), is evidently able to keep up with the increased rate of DXP production and maintain DXP levels constant.…”

Section: Main Textmentioning

confidence: 99%

Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway

Evans,

Xu,

Bergman

et al. 2024

Preprint

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Ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) produces pyruvate in the chloroplast through beta-elimination of the aci-carbanion intermediate. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid, and branched chain amino acid biosynthesis in photosynthetically active tissue. 13C labeling studies of whole Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate (PEP) to serve as precursor. Low oxygen stimulates rubisco carboxylase activity and increased pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis. Metabolome analysis of mutants defective in PEP or pyruvate import further supported rubisco as the main source of pyruvate in chloroplasts. Rubisco beta-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle (CBC). These insights solve the so-called pyruvate paradox, improve the fit of metabolic models for central metabolism, and connect the MEP pathway directly to carbon assimilation.

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MEP pathway products allosterically promote monomerization of deoxy-D-xylulose-5-phosphate synthase to feedback-regulate their supply

Cited by 17 publications

References 46 publications

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

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

Development ofCorynebacterium glutamicumas a monoterpene production platform

Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway

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