A synthetic mevalonate operon for studying the methyl erythritol phosphate pathway

Every organism in nature requires isoprenoid compounds to survive. Two five-carbon building blocks, isopententyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), are used in the biosynthesis of all isoprenoids. Nature has devised two distinct pathways for their formation. In eukaryea and archaea, the mevalonate (MVA) pathway is used exclusively. Plants utilize the MVA pathway to synthesize cytosolic isoprenoids and some gram positive bacteria also use this pathway. Gram negative bacteria, and most gram positive bacteria, biosynthesize isoprenoids by the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway. Plastid derived isoprenoids in plants are also formed by the MEP pathway. Due to the observed distribution of the two pathways, the rather newly identified and significantly less studied MEP pathway is an ideal target for the development of anti-infective agents.; The introductory chapter of this thesis provides a brief background of isoprenoids, outlines the two pathways and describes the steps involved. Chapter 2 describes the construction of a synthetic operon to be utilized as a tool for the study of the MEP pathway. The synthetic operon consists of three yeast genes responsible for converting MVA to IPP, in effect introducing the MVA pathway into an organism that previously lacked this pathway. The MEP pathway was disrupted in Salmonella typhimurium by inserting the synthetic operon into the gene encoding deoxylulose synthase (dxs ). Chapter 3 describes the identification of the srlE gene product as being responsible for the transport and phosphorylation of exogenously supplied methyl erythritol (ME). The gene was discovered during random mutagenesis experiments and was verified by feeding experiments utilizing deuterium and 14C labeled ME. The identification of a bifunctional ispDF protein encoding two genes involved in the MEP pathway is described in Chapter 4. The individual activities associated with ispD and ispE proteins were verified by incubating the ispDF protein with radiolabeled substrates and analysis of the products by LC/MS. Chapter 5 explores the nature of the reactions catalyzed by FPP synthase. The rates of catalysis observed for geranyl diphosphate (GPP) and 3-desmethyl GPP (dGPP) were compared and the data support a mechanism involving the formation of a carbocationic intermediate.