Structure-function correlation in metalloenzymes: Aristolochene synthase and arginase I

Aristolochene synthase is a Mg2+-dependent enzyme that catalyzes the cyclization of farnesyl diphosphate to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. The 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. In both conformations, but especially in the closed conformation, the active site contour is complementary in shape to aristolochene, and a catalytic function is proposed for the pyrophosphate anion.;The 2.1 A resolution crystal structure of aristolochene synthase complexed with substrate farnesyl diphopshate (FPP) reveals the binding of intact FPP to monomers A-C, and the binding of PPi and Mg 2+3 to monomer D of the tetramer. The 1.89 A resolution structure of aristolochene synthase complexed with the reactive substrate analogue 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg2+B accommodating the binding of this analogue only in monomer D. All monomers adopt "open" active site conformations in these complexes. Finally, the 2.4 A resolution structure of aristolochene synthase complexed with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the "open" conformation, and the binding of PPi, Mg2+ B, and Mg2+C to monomer D in the "closed" conformation. Taken together, these structures provide 12 independent "snapshots" of ligand binding to aristolochene synthase that suggest a specific metal ion binding sequence for catalysis: Mg2+B binds first to govern the initial molecular recognition of FPP; Mg 2+C then binds to trigger the "open" → "closed" conformational transition of the active site; finally, Mg2+A binds to complete the trinuclear magnesium cluster, which initiates the cyclization cascade.