Abstract
The essential enzyme 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECP) synthase, found in most eubacteria and the apicomplexan parasites, participates in isoprenoid-precursor biosynthesis and is a validated target for the development of broad-spectrum antimicrobial drugs. The structure and mechanism of the enzyme have been elucidated and the recent exciting finding that the enzyme actually binds diphosphate-containing isoprenoids at the interface formed by the three subunits that constitute the active protein suggests the possibility of feedback regulation of MECP synthase. To investigate such a possibility, a form of the enzyme was sought that did not bind these ligands but which would retain the quaternary structure necessary to create the active site. Two amino acids, Arg142 and Glu144, in Escherichia coli MECP synthase were identified as contributing to ligand binding. Glu144 interacts directly with Arg142 and positions the basic residue to form two hydrogen bonds with the terminal phosphate group of the isoprenoid diphosphate ligand. This association occurs at the trimer interface and three of these arginines interact with the ligand phosphate group. A dual mutation was designed (Arg142 to methionine and Glu144 to leucine) to disrupt the electrostatic attractions between the enzyme and the phosphate group to investigate whether an enzyme without isoprenoid diphosphate could be obtained. A low-resolution crystal structure of the mutated MECP synthase Met142/Leu144 revealed that geranyl diphosphate was retained despite the removal of six hydrogen bonds normally formed with the enzyme. This indicates that these two hydrophilic residues on the surface of the enzyme are not major determinants of isoprenoid binding at the trimer interface but rather that hydrophobic interactions between the hydrocarbon tail and the core of the enzyme trimer dominate ligand binding.
Highlights
The enzyme 2C-methyl-d-erythritol-2,4-cyclodiphosphate (MECP) synthase catalyzes the fifth stage in the biosynthesis of isoprenoid precursors by the 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway
The sequential additions of isopentenyl diphosphate (IPP) to the allylic precursor are catalyzed by isoprenyl diphosphate synthases to give geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and longer chain products (Kellogg & Poulter, 1997; Leyes et al, 1999)
The loss of direct interactions between enzyme and ligand allows a greater degree of flexibility of the terminal phosphate group and this is reflected in an altered B-factor distribution in the ligand compared with the wildtype complex
Summary
The enzyme 2C-methyl-d-erythritol-2,4-cyclodiphosphate (MECP) synthase catalyzes the fifth stage in the biosynthesis of isoprenoid precursors by the 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. This biosynthetic route, sometimes referred to as the 2C-methyld-erythritol-4-phosphate (MEP) pathway (Eisenreich et al, 1998; Lichtenthaler, 1999), is present in many bacteria, the plastids of plants and in apicomplexan parasites. The sequential additions of IPP to the allylic precursor are catalyzed by isoprenyl diphosphate synthases to give geranyl diphosphate (GPP), farnesyl diphosphate (FPP) and longer chain products (Kellogg & Poulter, 1997; Leyes et al, 1999) These compounds serve as precursors for the biosynthesis of more complex natural products including sterols, dolichols, triterpenes, ubiquinones and plastoquinones (Sacchettini & Poulter, 1997)
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