Enzymatic characterization of Escherichia coli methylerythritol phosphate synthase and synthesis of methylerythritol phosphate pathway metabolites

The methylerythritol phosphate (MEP) pathway has been identified as a novel route to isoprenoid compounds operating in bacteria, algae, and plant chloroplasts. MEP synthase, which catalyzes the rearrangement/reduction of 1-deoxy-D-xylulose-5-phosphate (DXP) to form 2-C-methyl- D-erythritol-4-phosphate (MEP), is a key step in the pathway and an important pharmacological target.; Chapter 1 of this dissertation provides background information on the biosynthesis of isoprenoids and on the enzymes in the MEP pathway, with a focus on MEP synthase.; The chemical syntheses of the MEP pathway metabolites MEP and 4-diphosphocytidyl-2-C-methyl- D-erythritol (CDPME) are described in Chapter 2. MEP was readily prepared as the sodium salt or as the free acid from commercially available materials with overall yields of 27% (75:25 ex.) and 32% (78:22 ex.), respectively. CDPME was prepared in 40% yield from MEP using a recently developed carbohydrate-nucleotide diphosphate coupling technique.; In a second project described in Chapter 3, recombinant Escherichia coli MEP synthase was purified by chromatography on DE-52 and phenylsepharose, and its steady-state kinetic constants were determined at 37°C and pH = 7.6; k_cat = 116 ± s−1, K_MDXP = 115 ± 25 μM, and K_M NADPH = 0.5 ± 0.2 μM. Synthetic MEP was used to show that the rearrangement/reduction is reversible; K_eq37° = 45 ± 6 for DXP and MEP at 150 μM NADPH. The mechanism for substrate binding was examined using fosmidomycin and dihydro-NADPH as dead-end inhibitors. Dihydro-NADPH gave a competitive pattern against NADPH and a noncompetitive pattern against DXP. Fosmidomycin was an uncompetitive inhibitor against NADPH and gave a pattern representative of slow, tight binding competitive inhibition against DXP. These results are consistent with an ordered mechanism where NADPH binds before DXP; Chapter 4 documents the cloning and identification of a novel squalene synthase from the cyanobacterium Synechocystis sp. The protein lacks a region conserved in homologous eukaryotic proteins that encodes for a membrane-anchoring domain and is naturally soluble. The gene is toxic, however, and methylerythritol is an essential bacterial supplement for transformants. The use of methylerythritol as a supplement for expression of other isoprenoid genes is discussed.