Crystal structure of mevalonate 3,5-bisphosphate decarboxylase reveals insight into the evolution of decarboxylases in the mevalonate metabolic pathways.

Mevalonate 3,5-bisphosphate decarboxylase is involved in the recently discovered Thermoplasma-type mevalonate pathway. The enzyme catalyzes the elimination of the 3-phosphate group from mevalonate 3,5-bisphosphate as well as concomitant decarboxylation of the substrate. This entire reaction of the enzyme resembles the latter half-reactions of its homologs, diphosphomevalonate decarboxylase and phosphomevalonate decarboxylase, which also catalyze ATP-dependent phosphorylation of the 3-hydroxyl group of their substrates. However, the crystal structure of mevalonate 3,5-bisphosphate decarboxylase and the structural reasons of the difference between reactions catalyzed by the enzyme and its homologs are unknown. In this study, we determined the X-ray crystal structure of mevalonate 3,5-bisphosphate decarboxylase from Picrophilus torridus, a thermoacidophilic archaeon of the order Thermoplasmatales. Structural and mutational analysis demonstrated the importance of a conserved aspartate residue for enzyme activity. In addition, although crystallization was performed in the absence of substrate or ligands, residual electron density having the shape of a fatty acid was observed at a position overlapping the ATP-binding site of the homologous enzyme, diphosphomevalonate decarboxylase. This finding is in agreement with the expected evolutionary route from phosphomevalonate decarboxylase (ATP-dependent) to mevalonate 3,5-bisphosphate decarboxylase (ATP-independent) through the loss of kinase activity. We found that the binding of geranylgeranyl diphosphate, an intermediate of the archeal isoprenoid biosynthesis pathway, evoked significant activation of mevalonate 3,5-bisphosphate decarboxylase, and several mutations at the putative geranylgeranyl diphosphate-binding site impaired this activation, suggesting the physiological importance of ligand binding as well as a possible novel regulatory system employed by the Thermoplasma-type mevalonate pathway.

Synthesis of 1,2-Bis(2-aryl-1H-indol-3-yl)ethynes via 5-exo-Digonal Double Cyclization Reactions of 1,4-Bis(2-isocyanophenyl)buta-1,3-diyne with Aryl Grignard Reagents.

New π-conjugated 1,2-bis(2-aryl-1H-indol-3-yl)ethynes 1a-j having various substituents on the two aryl groups were efficiently synthesized via unusual 5-exo-digonal double isocyanide-acetylene cyclization reactions of 1,4-bis(2-isocyanophenyl)buta-1,3-diyne 3 and aryl Grignard reagents (R-MgBr, R = C6H5 (1a), 4-H3CC6H4 (1b), 2-H3CC6H4 (1c), 3-MeOC6H4 (1d), 3-(CH3)2NC6H4 (1e), 4-F3CC6H4 (1f), 4-FC6H4 (1g), 3-FC6H4 (1h), 4-PhOC6H4 (1i), and 2-Naph (1j)) in 19-85% yields. The UV-vis spectra were rationalized in detail using time-dependent DFT and single point calculations. The fluorescence emission peaks for 1a-j were observed at around 450 nm. Especially for 1f and 1j, those spectra displayed broad emission bands and relatively large Stokes shifts (3977-4503 cm-1), indicating the contribution of an intramolecular charge transfer. The absolute quantum yields (0.50-0.62) of 1a-j were higher than those of parent 8 (0.19) and 2-phenyl-1H-indole (0.11). The electrochemical features for 1a-j were investigated by cyclic voltammetry. The frontier molecular orbital levels for 1a-j were estimated based on the combination of oxidation potentials, UV-vis, and DFT calculated data. The structural property of 1,2-bis(2-phenyl-1H-indol-3-yl)ethyne 1a was characterized by several spectroscopic methods and finally determined by X-ray analysis of a single crystal of 1a recrystallized from ethyl acetate. The structural features of 1a-j were also supported by DFT calculations.