Structure and Mechanism of 2-C-Methyl-d-erythritol 2,4-Cyclodiphosphate Synthase

The enzyme 2-C-methyl-d-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-d-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-Å resolution. The first structure contains a bound Mn²⁺ cation, and the second structure contains CMP, MECDP, and Mn²⁺. The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn²⁺, sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp⁸, His¹⁰, and His⁴² occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn²⁺ recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.