Abstract
The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D. The 1.89-A resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg(2+)(B)accompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-A resolution structure of the complex 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 PP(i), Mg(2+)(B), and Mg(2+)(C) to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent "snapshots" of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.
Highlights
Terpenes found in numerous plants, bacteria, and fungi [1, 2]
To study the conformational control of farnesyl diphosphate (FPP) in the active site of aristolochene synthase from A. terreus, we report the structures of crystalline complexes
Aristolochene Synthase1⁄7Substrate Complexes conformations and substrate conformations appear to be linked to differences in metal binding, analysis of which suggests a possible sequence for metal ion binding and conformational changes required for catalysis
Summary
Terpenes found in numerous plants, bacteria, and fungi [1, 2]. The cyclization of FPP is catalyzed by a sesquiterpene cyclase that utilizes a trinuclear magnesium cluster to trigger the departure of the pyrophosphate (PPi) leaving group, thereby forming an allylic carbocation that typically reacts with one of the remaining bonds of the substrate [3,4,5,6,7]. Aristolochene synthase from Aspergillus terreus is a sesquiterpene cyclase that catalyzes the cyclization of FPP to form aristolochene (Fig. 1a), the parent hydrocarbon of a large group of fungal toxins such as gigantenone, PR-toxin, and bipolaroxin [8]. It is likely that the diphosphate group of FPP makes identical metal coordination and hydrogen bond interactions in the Michaelis complex, i.e. the complex between the enzyme and the productively bound substrate that immediately precedes the initiation of the cyclization cascade. The active site of aristolochene synthase from A. terreus serves as a high fidelity template that fixes FPP in a single, productive conformation in the Michaelis complex; otherwise, aberrant cyclization products would result. To study the conformational control of FPP in the active site of aristolochene synthase from A. terreus, we report the structures of crystalline complexes. Aristolochene Synthase1⁄7Substrate Complexes conformations and substrate conformations appear to be linked to differences in metal binding, analysis of which suggests a possible sequence for metal ion binding and conformational changes required for catalysis
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