Báo cáo khoa học: Isoprenoid biosynthesis via the methylerythritol phosphate pathway Mechanistic investigations of the 1-deoxy-D-xylulose 5-phosphate reductoisomerase

Isoprenoid Biosynthesis Via the Methylerythritol Phosphate Pathway: Mechanistic Investigations of the 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase

Authors: Jean-François Hoeffler, Denis Tritsch, Catherine Grosdemange-Billiard, Michel Rohmer

Affiliation: Université Louis Pasteur, CNRS, Institut Le Bel, Strasbourg, France

Field: Biochemistry, Molecular Biology

Document Content:

This study investigates the enzymatic mechanism of 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), a key enzyme in the mevalonate-independent pathway for isoprenoid biosynthesis in various organisms. The research focuses on understanding the role of NADPH, the potential involvement of intermediates, and the reversibility of the DXR-catalyzed reaction. Experimental evidence is presented regarding the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) to 2-C-methyl-D-erythritol 4-phosphate (MEP), including the proposed intermediate 2-C-methyl-D-erythrose 4-phosphate. The study utilizes isotopic labeling and various biochemical techniques to elucidate the reaction steps and the influence of cofactors and metal ions. Furthermore, the reversibility of the DXR reaction, leading to the formation of DXP from MEP, is demonstrated, and the equilibrium constant is determined. The findings contribute to a deeper understanding of isoprenoid biosynthesis and have implications for the design of potential inhibitors.

Detailed Table of Contents:

• Introduction to Isoprenoid Biosynthesis and the MEP Pathway
• The Role of 1-Deoxy-D-xylulose 5-Phosphate Reductoisomerase (DXR)
• Materials and Methods
• Synthesis of Key Intermediates and Analogues
• Purification of His-tagged Deoxyxylulose 5-Phosphate Reductoisomerase
• Enzymatic Activity Assays
• Kinetic Studies
• Results and Discussion:
• 2-C-Methyl-D-erythrose 4-Phosphate as an Intermediate in the DXR-catalyzed Reaction
• Formation of 2-C-Methyl-D-erythrose 4-Phosphate from DXP: a-ketol rearrangement vs. retro-aldolization/aldolization
• Reversibility of the DXR-catalyzed Reaction: Formation of DXP 3 from MEP 5
• Determination of the Apparent Equilibrium Constant of the DXR Reaction
• 13C-NMR Study of the Rearrangement Reaction of H-DXR
• Acknowledgements
• References