Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which...Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which are produced by the methylerythritol 4-phosphate(MEP)pathway in bacteria and plant plastids.It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase(DXS),a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway.Here we provide experimental insights intotheunderlyingmechanism.Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro,causing a size shift.In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer.Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells.Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply,whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance(e.g.,by a blockage in their conversion to downstream isoprenoids).Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.展开更多
基金funded by grants from the Spanish MCIN/AEI/10.13039/501100011033European ERDF/FEDER,NextGeneration EU/PRTR and PRIMA programs(PID2020-115810GB-I00+3 种基金UToPIQ-PCI2021-121941 to M.R.-C.and BFU2016-78232-P to A.V.-C.).M.R.-C.is also supported by CSIC(202040E299)Generalitat Valenciana(PROMETEU/2021/056).R.K.and E.E.K.B.conducted the metabolite analysis at the Joint BioEnergy Institute(http://www.jbei.org),supported by the US Department of Energy,Office of Science,Office of Biological and Environmental Research under contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the US Department of Energy.J.P.-Gwas supported by a Marie Curie International Outgoing Fellowship within the EC-FP7 Program(project 627639)X.D.was supported by the China Scholarship Council and D.O.-A.by an MCIN/AEI/fellowship(BES-2017-080739).
文摘Isoprenoids are a very large and diverse family of metabolites required by all living organisms.All isoprenoids derive fromthe double-bond isomers isopentenyl diphosphate(IPP)and dimethylallyl diphosphate(DMAPP),which are produced by the methylerythritol 4-phosphate(MEP)pathway in bacteria and plant plastids.It has been reported that IPP and DMAPP feedback-regulate the activity of deoxyxylulose 5-phosphate synthase(DXS),a dimeric enzyme that catalyzes the main flux-controlling step of the MEP pathway.Here we provide experimental insights intotheunderlyingmechanism.Isothermal titration calorimetry and dynamic light scattering approaches showed that IPP and DMAPP can allosterically bind to DXS in vitro,causing a size shift.In silico ligand binding site analysis and docking calculations identified a potential allosteric site in the contact region between the two monomers of the active DXS dimer.Modulation of IPP and DMAPP contents in vivo followed by immunoblot analyses confirmed that high IPP/DMAPP levels resulted in monomerization and eventual aggregation of the enzyme in bacterial and plant cells.Loss of the enzymatically active dimeric conformation allows a fast and reversible reduction of DXS activity in response to a sudden increase or decrease in IPP/DMAPP supply,whereas aggregation and subsequent removal of monomers that would otherwise be available for dimerization appears to be a more drastic response in the case of persistent IPP/DMAPP overabundance(e.g.,by a blockage in their conversion to downstream isoprenoids).Our results represent an important step toward understanding the regulation of the MEP pathway and rational design of biotechnological endeavors aimed at increasing isoprenoid contents in microbial and plant systems.
