The 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway is an important pathway for the biosynthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), the precursors of isoprenoids. Isoprenoids are ubiquitous natural products present in all different forms of life and have a wide variety of structures and functions. Some isoprenoids are involved in primary metabolic processes like photosynthesis, respiration, regulation of growth and development whereas many others have important roles in secondary metabolism. In addition, isoprenoids have numerous commercial applications as flavor and fragrance molecules, drugs, pigments, natural polymers, agrichemicals, cosmetics, biofuels, etc. Isoprene, the smallest member of the isoprenoid family, has different adverse effects on atmospheric chemistry. Despite having diverse functions, all isoprenoids are structurally based on C5 isoprenoid units. It was believed for a long time that the mevalonate (MVA) pathway is the only route for the biosynthesis of IDP. In early 1990s, it was discovered that an alternative (MEP) pathway exists for the biosynthesis of both IDP and DMADP. Soon after its discovery, the various enzymes and metabolites involved in this pathway were elucidated. However, not much was known about the metabolic regulation of this pathway. Considering the numerous applications of isoprenoids, it was important to understand the metabolic regulation of the MEP pathway. Earlier studies suggested that DXS might play an important role in the regulation of the MEP pathway. The research work presented in this dissertation mainly involves the study of the metabolic regulation of this pathway by focusing on the kinetic behavior of 1-deoxy-D-xylulose-5-phosphate synthase (DXS). A liquid chromatography-tandem mass spectrometry (LC-MS/MS) based assay was developed for DXS to study its kinetics in presence of different metabolites of the MEP pathway. It was observed that recombinant DXS from Populus trichocarpa (PtDXS) is feedback-inhibited by IDP and DMADP, the two end products of this pathway. Mechanistic studies of this inhibition showed that both IDP and DMADP compete with thiamin diphosphate (ThDP) for binding at the active site of the enzyme. Feedback regulation of DXS plays an important role in controlling the carbon flux through this pathway and thus constitutes a significant regulatory mechanism of this pathway. A modified PtDXS, which would exhibit reduced binding affinity for IDP/DMADP and thereby relieving the feedback inhibition partially or completely, would be important for biotechnological uses. Site-directed mutagenesis was used to engineer an improved PtDXS that has reduced affinity for IDP and DMADP. This engineered PtDXS was also shown to have higher Km for ThDP than the WT enzyme. Therefore, this mutant of PtDXS would be important for biotechnological applications if high concentration of ThDP is maintained.
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