| It has been widely accepted for many decades that isoprenoid biosynthesis occurs ubiquitously via the mevalonate (MVA) pathway. More recently a second independent route for isoprenoid biosynthesis was discovered in bacteria, green algae, and plants. This newly discovered pathway, the methylerythritol phosphate (MEP) pathway, begins with the condensation of pyruvate and D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). DXP synthase catalyzes this first enzymatic step of the MEP pathway. The two distinct routes for isoprenoid biosynthesis converge at the intermediates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). All isoprenoids are derived from IPP and DMAPP, which represent the fundamental five-carbon building blocks. Chain elongation reactions then proceed to form a pool of linear allylic diphosphates, many which serve as branch points for production of specific isoprenoids, including prenylated proteins. Two enzymes that catalyze protein prenylation are protein farnesyltransferase (PFTase) and protein geranylgeranyltranferase type I (PGGTase I). Both enzymes catalyze the transfer of a hydrophobic C15 farnesyl or C20 geranylgeranyl group, respectively, to a protein substrate that requires this modification for membrane association.; Chapter 1 of this dissertation provides general background information about isoprenoid biosynthesis, thiamine diphosphate (TPP)-dependent enzymes, including DXP synthase, and the posttranslational modification of proteins, including protein prenylation. Chapter 2 describes a project in which two DXP synthases from Rhodobacter capsulatus (R. capsulatus ) were produced in Escherichia coli (E. coli), purified, and characterized. To determine steady-state kinetic constants and divalent metal requirements, a new enzyme assay was developed which measures the incorporation of 14C into [2-14C]DXP or [2-14C]DXS from [2-14C]pyruvate. Chapter 3 details a project in which the kinetic mechanism and substrate binding of DXP synthase were investigated. Steady-state kinetic studies, inhibition studies, and radiolabeled 14CO2-trapping experiments provided data consistent with an ordered sequential mechanism in which both substrates must bind to the enzyme active site for catalysis to occur efficiently. In a fourth project detailed in Chapter 4, a series of substrate diphosphate analogs of farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) were investigated with PFTase and PGGTase I, respectively. All of the analogs were determined to be competitive inhibitors with respect to the natural substrates, FPP or GGPP, and several were also alternative substrates. |
