title: Feedback inhibition of deoxy-D-xylulose-5-phosphate synthase regulates the methylerythritol 4-phosphate pathway.
abstract: The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway leads to the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors for isoprene and higher isoprenoids. Isoprene has significant effects on atmospheric chemistry, whereas other isoprenoids have diverse roles ranging from various biological processes to applications in commercial uses. Understanding the metabolic regulation of the MEP pathway is important considering the numerous applications of this pathway. The 1-deoxy-D-xylulose-5-phosphate synthase (DXS) enzyme was cloned from Populus trichocarpa, and the recombinant protein (PtDXS) was purified from Escherichia coli. The steady-state kinetic parameters were measured by a coupled enzyme assay. An LC-MS/MS-based assay involving the direct quantification of the end product of the enzymatic reaction, 1-deoxy-D-xylulose 5-phosphate (DXP), was developed. The effect of different metabolites of the MEP pathway on PtDXS activity was tested. PtDXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of PtDXS in complex with thiamine pyrophosphate and Mg(2+) was built by homology modeling and refined by molecular dynamics simulations. The refined structure was used to model the binding of IDP and DMADP and indicated that IDP and DMADP might bind with the enzyme in a manner very similar to the binding of thiamine pyrophosphate. The feedback inhibition of PtDXS by IDP and DMADP constitutes an important mechanism of metabolic regulation of the MEP pathway and indicates that thiamine pyrophosphate-dependent enzymes may often be affected by IDP and DMADP.
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Aparajita
authors.initials: A
authors.lastname: Banerjee
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Yan
authors.initials: Y
authors.lastname: Wu
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Rahul
authors.initials: R
authors.lastname: Banerjee
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824.
authors.forename: Yue
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824. Electronic address: yanh@msu.edu.
authors.forename: Honggao
authors.initials: H
authors.lastname: Yan
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan 48824. Electronic address: tsharkey@msu.edu.
authors.forename: Thomas D
authors.initials: TD
authors.lastname: Sharkey
mesh_terms: D004896:Erythritol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli; D045782:Hemiterpenes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D032107:Populus / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013403:Sugar Phosphates / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013835:Thiamine Pyrophosphate / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014166:Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: C114232:2-C-methylerythritol 4-phosphate; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010940:Plant Proteins; D011994:Recombinant Proteins; D013403:Sugar Phosphates; C004809:isopentenyl pyrophosphate; C043060:3,3-dimethylallyl pyrophosphate; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D013835:Thiamine Pyrophosphate; D004896:Erythritol
keywords: Deoxyxylulose-5-phosphate Synthase; Enzyme Inhibitors; Enzyme Structure; Isoprene; Isoprenoid; Methylerythritol Pathway; Plant Biochemistry; Thiamine; Thiamine Diphosphate