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Feedback Inhibition of Deoxy-d-xylulose-5-phosphate Synthase Regulates the Methylerythritol 4-Phosphate Pathway

2013; Elsevier BV; Volume: 288; Issue: 23 Linguagem: Inglês

10.1074/jbc.m113.464636

1083-351X

Aparajita Banerjee, Yan Wu, R. Banerjee, Yue Li, Honggao Yan, Thomas D. Sharkey,

Lipid metabolism and biosynthesis

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 ( Pt DXS) 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 Pt DXS activity was tested. Pt DXS was inhibited by IDP and DMADP. Both of these metabolites compete with thiamine pyrophosphate for binding with the enzyme. An atomic structural model of Pt DXS 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 Pt DXS 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. Background: The methylerythritol phosphate (MEP) pathway is required for the biosynthesis of plastid-derived isoprenoids from plants. Results: Deoxyxylulose-5-phosphate synthase (DXS) was cloned from Populus trichocarpa , and metabolic regulation was tested. Conclusion: Both isopentenyl diphosphate and dimethylallyl diphosphate inhibit DXS by competing with thiamine pyrophosphate. Significance: Prediction of isoprene emission from trees and bioengineering of MEP pathway will be aided by these results.