Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Murkin, Andrew S.; Manning, Kathryn A.; Kholodar, Svetlana A.

doi:10.1016/j.bioorg.2014.06.001

Cited by 31 publications

(26 citation statements)

References 125 publications

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“…DXR performs a multistep reaction that proceeds through retro-aldol/aldol isomerization of the ketone DXP to the branched aldehyde intermediate 2- C -methyl- d -erythrose 4-phosphate (MEsP), followed by NADPH-dependent reduction to form 2- C -methyl- d -erythritol 4-phosphate (MEP) (Scheme 1 ). 17 Importantly, a phosphodianion group is critical for substrate turnover 18 , 19 and for inhibition by the antibiotics fosmidomycin and FR-900098. In our previous study, we demonstrated that 1-deoxy- l -erythrulose (DE), a DXP analogue lacking the terminal phosphorylmethyl group, can be converted by Mycobacterium tuberculosis DXR ( Mt DXR) to the corresponding MEP analogue 2- C -methylglycerol (2MG) with a k cat / K m that is 10 6 lower than that for DXP.…”

Section: Introductionmentioning

confidence: 99%

The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces

et al. 2015

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Several mechanistically unrelated enzymes utilize the binding energy of their substrate’s nonreacting phosphoryl group to accelerate catalysis. Evidence for the involvement of the phosphodianion in transition state formation has come from reactions of the substrate in pieces, in which reaction of a truncated substrate lacking its phosphorylmethyl group is activated by inorganic phosphite. What has remained unknown until now is how the phosphodianion group influences the reaction energetics at different points along the reaction coordinate. 1-Deoxy-d-xylulose-5-phosphate (DXP) reductoisomerase (DXR), which catalyzes the isomerization of DXP to 2-C-methyl-d-erythrose 4-phosphate (MEsP) and subsequent NADPH-dependent reduction, presents a unique opportunity to address this concern. Previously, we have reported the effect of covalently linked phosphate on the energetics of DXP turnover. Through the use of chemically synthesized MEsP and its phosphate-truncated analogue, 2-C-methyl-d-glyceraldehyde, the current study revealed a loss of 6.1 kcal/mol of kinetic barrier stabilization upon truncation, of which 4.4 kcal/mol was regained in the presence of phosphite dianion. The activating effect of phosphite was accompanied by apparent tightening of its interactions within the active site at the intermediate stage of the reaction, suggesting a role of the phosphodianion in disfavoring intermediate release and in modulation of the on-enzyme isomerization equilibrium. The results of kinetic isotope effect and structural studies indicate rate limitation by physical steps when the covalent linkage is severed. These striking differences in the energetics of the natural reaction and the reactions in pieces provide a deeper insight into the contribution of enzyme–phosphodianion interactions to the reaction coordinate.

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Section: Introductionmentioning

confidence: 99%

The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces

et al. 2015

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“…After decarboxylation of Py has taken place, DXPS catalyzes the subsequent carboligation to D-glyceraldehyde 3-phosphate (GAP) forming DXP, reminiscent of the reactions of essential mammalian enzymes, such as transketolase. Previous mechanistic and structural studies have uncovered several distinguishing features of DXPS among the superfamily of ThDP-dependent enzymes (12)(13)(14)(15)(16)(17). Unlike the classical ping-pong mechanism exhibited by other ThDP-dependent enzymes, DXPS catalyzes a random sequential, preferred order mechanism, which is unprecedented in ThDP-dependent enzymology (13)(14)(15)(16)(17).…”

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confidence: 99%

“…Previous mechanistic and structural studies have uncovered several distinguishing features of DXPS among the superfamily of ThDP-dependent enzymes (12)(13)(14)(15)(16)(17). Unlike the classical ping-pong mechanism exhibited by other ThDP-dependent enzymes, DXPS catalyzes a random sequential, preferred order mechanism, which is unprecedented in ThDP-dependent enzymology (13)(14)(15)(16)(17). The unique feature of DXPS is a stabilization of the enzyme-bound tetrahedral predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), formed on Py binding ( Fig.…”

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confidence: 99%

Conformational dynamics of 1-deoxy- d -xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS

Zhou

Yang

DeColli

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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The enzyme 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) is a key enzyme in the methylerythritol 4-phosphate pathway and is a target for the development of antibiotics, herbicides, and antimalarial drugs. DXPS catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP), a branch point metabolite in isoprenoid biosynthesis, and is also used in the biosynthesis of thiamin (vitamin B) and pyridoxal (vitamin B). Previously, we found that DXPS is unique among the superfamily of thiamin diphosphate (ThDP)-dependent enzymes in stabilizing the predecarboxylation intermediate, C2-alpha-lactyl-thiamin diphosphate (LThDP), which has subsequent decarboxylation that is triggered by d-glyceraldehyde 3-phosphate (GAP). Herein, we applied hydrogen-deuterium (H/D) exchange MS (HDX-MS) of full-length DXPS to provide a snapshot of the conformational dynamics of this enzyme, leading to the following conclusions. () The high sequence coverage of DXPS allowed us to monitor structural changes throughout the entire enzyme, including two segments (spanning residues 183-238 and 292-317) not observed by X-ray crystallography. () Three regions of DXPS (spanning residues 42-58, 183-199, and 278-298) near the active center displayed both EX1 (monomolecular) and EX2 (bimolecuar) H/D exchange (HDX) kinetic behavior in both ligand-free and ligand-bound states. All other peptides behaved according to the common EX2 kinetic mechanism. () The observation of conformational changes on DXPS provides support for the role of conformational dynamics in the DXPS mechanism: The closed conformation of DXPS is critical for stabilization of LThDP, whereas addition of GAP converts DXPS to the open conformation that coincides with decarboxylation of LThDP and DXP release.

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“…As little is known about the multiple levels of the MEP pathway regulation in S. elongatus PCC 7942, it is not clear why overexpression of Dxr negatively affected production. Fine-tuning gene expression, such as with the RBS calculator [ 45 ] to fine-tune production or by adding protein degradation tags [ 46 ] to Dxr to fine-tune degradation, may be required to balance the gene expression of Dxr for the MEP pathway flux, because E. coli Dxr has lower K m value (30 µM) and faster k cat (100 s −1 ) than the cyanobacterial enzyme ( K m of 134 µM and k cat of 5 s −1 ) [ 47 ].…”

Section: Resultsmentioning

confidence: 99%

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria

Choi

Lee

Choi

et al. 2016

Biotechnol Biofuels

100

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BackgroundMetabolic engineering of cyanobacteria has enabled photosynthetic conversion of CO2 to value-added chemicals as bio-solar cell factories. However, the production levels of isoprenoids in engineered cyanobacteria were quite low, compared to other microbial hosts. Therefore, modular optimization of multiple gene expressions for metabolic engineering of cyanobacteria is required for the production of farnesyl diphosphate-derived isoprenoids from CO2.ResultsHere, we engineered Synechococcus elongatus PCC 7942 with modular metabolic pathways consisting of the methylerythritol phosphate pathway enzymes and the amorphadiene synthase for production of amorpha-4,11-diene, resulting in significantly increased levels (23-fold) of amorpha-4,11-diene (19.8 mg/L) in the best strain relative to a parental strain. Replacing amorphadiene synthase with squalene synthase led to the synthesis of a high amount of squalene (4.98 mg/L/OD730). Overexpression of farnesyl diphosphate synthase is the most critical factor for the significant production, whereas overexpression of 1-deoxy-d-xylulose 5-phosphate reductase is detrimental to the cell growth and the production. Additionally, the cyanobacterial growth inhibition was alleviated by expressing a terpene synthase in S. elongatus PCC 7942 strain with the optimized MEP pathway only (SeHL33).ConclusionsThis is the first demonstration of photosynthetic production of amorpha-4,11-diene from CO2 in cyanobacteria and production of squalene in S. elongatus PCC 7942. Our optimized modular OverMEP strain (SeHL33) with either co-expression of ADS or SQS demonstrated the highest production levels of amorpha-4,11-diene and squalene, which could expand the list of farnesyl diphosphate-derived isoprenoids from CO2 as bio-solar cell factories.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0617-8) contains supplementary material, which is available to authorized users.

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Mechanism and inhibition of 1-deoxy-d-xylulose-5-phosphate reductoisomerase

Cited by 31 publications

References 125 publications

The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces

The Role of Phosphate in a Multistep Enzymatic Reaction: Reactions of the Substrate and Intermediate in Pieces

Conformational dynamics of 1-deoxy- d -xylulose 5-phosphate synthase on ligand binding revealed by H/D exchange MS

Photosynthetic conversion of CO2 to farnesyl diphosphate-derived phytochemicals (amorpha-4,11-diene and squalene) by engineered cyanobacteria

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