Integrated experimental and computational studies of 1-deoxy-D-xylulose 5-phosphate reductoisomerase

Michigan State University, East Lansing MI

Investigators

Abstract

Terpenoids are the most diverse, largest, single family of compounds found in nature. They play vital roles in all living organisms, and have a variety of applications in agriculture, nutrition, and medicine. The building blocks for the biosynthesis of terpenoids are made in nature by one of two biosynthetic pathways. The recently discovered MEP pathway is the pathway for the biosynthesis of the building blocks for terpenoids in most microorganisms, apicoplasts of some protozoa, and plastids of plants. 1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR) catalyzes the biosynthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP), the committing step of the MEP pathway. While there is rich structural information on DXR, the chemical mechanism of the DXR-catalyzed reaction remains unresolved and the transition state of the reaction is unknown. The proposed project is a combined experimental and computational study aimed at addressing these most challenging issues of this important enzyme. It involves the simultaneous measurement of multiple 13C kinetic isotope effects (KIE) on the DXP isomerization by a novel NMR-based method and combined quantum mechanics/molecular mechanics molecular dynamics simulation and 13C KIE computation. Furthermore, the transition state stabilization by DXR will be determined by site-directed mutagenesis and biochemical, X-ray crystallographic, and computational analysis of the site-directed mutant enzymes. The proposed research is not only of fundamental significance for the understanding of DXR catalysis but also valuable for the development of antibiotics, antimalarial drugs, and herbicides targeting DXR. It will also provide an outstanding opportunity for multidisciplinary research training of both graduate and undergraduate students. The results of the proposed research will be incorporated into protein biochemistry and computational chemistry courses taught by the PI and the co-PI at two institutions and will significantly enhance these courses and benefit students taking the courses for many years to come. This project is a study by a combination of modern experimental and computational methodologies on 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the most important enzyme in a pathway for the biosynthesis of the building blocks for terpenoids. This pathway is absent in human and animals but essential for most microorganisms, some protozoa, and plants. The research is not only of fundamental significance but also valuable for the development of antibiotics, antimalarial drugs, and herbicides targeting this enzyme. It will also provide an outstanding opportunity for multidisciplinary research training of both graduate and undergraduate students.