The mevalonate pathway produces isopentenyl diphosphate (IPP), a building block for polyisoprenoid synthesis, and is a crucial pathway for growth of the human bacterial pathogen The final enzyme in this pathway, mevalonate diphosphate decarboxylase (MDD), acts on mevalonate diphosphate (MVAPP) to produce IPP while consuming ATP. This essential enzyme has been suggested as a therapeutic target for the treatment of drug-resistant bacterial infections. Here, we report functional and structural studies on the mevalonate diphosphate decarboxylase from (MDD). The MDD crystal structure in complex with ATP (MDD-ATP) revealed that the phosphate-binding loop (amino acids 97-105) is not involved in ATP binding and that the phosphate tail of ATP in this structure is in an outward-facing position pointing away from the active site. This suggested that binding of MDD to MVAPP is necessary to guide ATP into a catalytically favorable position. Enzymology experiments show that the MDD performs a sequential ordered bi-substrate reaction with MVAPP as the first substrate, consistent with the isothermal titration calorimetry (ITC) experiments. On the basis of ITC results, we propose that this initial prerequisite binding of MVAPP enhances ATP binding. In summary, our findings reveal a substrate-induced substrate-binding event that occurs during the MDD-catalyzed reaction. The disengagement of the phosphate-binding loop concomitant with the alternative ATP-binding configuration may provide the structural basis for antimicrobial design against these pathogenic enterococci.

Mevalonate diphosphate decarboxylases (MDDs) catalyze the ATP-dependent-Mg 2+decarboxylation of mevalonate-5-diphosphate (MVAPP) to produce isopentenyl diphosphate (IPP), which is essential in both eukaryotes and prokaryotes for polyisoprenoid synthesis. The substrates, MVAPP and ATP, have been shown to bind sequentially to MDD. Here we report crystals in which the enzyme remains active, allowing the visualization of conformational changes in Enterococcus faecalis MDD that describe sequential steps in an induced fit enzymatic reaction. Initial binding of MVAPP modulates the ATP binding pocket with a large loop movement. Upon ATP binding, a phosphate binding loop bends over the active site to recognize ATP and bring the molecules to their catalytically favored configuration. Positioned substrates then can chelate two Mg 2+ ions for the two steps of the reaction. Closure of the active site entrance brings a conserved lysine to trigger dissociative phosphoryl transfer of γ-phosphate from ATP to MVAPP, followed by the production of IPP.