Mechanistic Insights from the Binding of Substrate and Carbocation Intermediate Analogues to Aristolochene Synthase

Abstract

Aristolochene synthase, a metal-dependent sesquiterpene cyclase from Aspergillus terreus, catalyzes the ionization-dependent cyclization of farnesyl diphosphate (FPP) to form the bicyclic eremophilane (+)-aristolochene with perfect structural and stereochemical precision. Here, we report the X-ray crystal structure of aristolochene synthase complexed with three Mg2+ ions and the unreactive substrate analogue farnesyl-S-thiolodiphosphate (FSPP), showing that the substrate diphosphate group is anchored by metal coordination and hydrogen bond interactions identical to those previously observed in the complex with three Mg2+ ions and inorganic pyrophosphate (PPi). Moreover, the binding conformation of FSPP directly mimics that expected for productively bound FPP, with the exception of the precise alignment of the C-S bond with regard to the C10-C11 π system that would be required for C1-C10 bond formation in the first step of catalysis. We also report crystal structures of aristolochene synthase complexed with Mg2+3-PPi and ammonium or iminium analogues of bicyclic carbocation intermediates proposed for the natural cyclization cascade. Various binding orientations are observed for these bicyclic analogues, and these orientations appear to be driven by favorable electrostatic interactions between the positively charged ammonium group of the analogue and the negatively charged PPi anion. Surprisingly, the active site is sufficiently flexible to accommodate analogues with partially or completely incorrect stereochemistry. Although this permissiveness in binding is unanticipated, based on the stereochemical precision of catalysis that leads exclusively to the (+)-aristolochene stereoisomer, it suggests the ability of the active site to enable controlled reorientation of intermediates during the cyclization cascade. Taken together, these structures illuminate important aspects of the catalytic mechanism.

Identification of a Fungal 1,8-Cineole Synthase from Hypoxylon sp. with Specificity Determinants in Common with the Plant Synthases

“…These models reveal several features in the active site pocket that may influence enzyme specificity and 1,8-cineole catalysis. In substrate-bound structures of A. terreus aristolochene synthase, the FPP substrate C12 and C13 carbons are positioned near the NSE motif-containing ␣-helix (62). Near this position, Leu-209 in the aristolochene synthase is replaced with the bulkier aromatic Phe-267 in Hyp3, which may interfere with the FPP substrate (Fig.…”

Enzymatic synthesis of natural (+)-aristolochene from a non-natural substrate

“…1). Docking of the constrained structures of 3 and 13 in the (highly homologous) closed active site of aristolochene synthase from A. terreus 35 (Fig. 2) demonstrates that both fit in the active site pocket in similar conformations and in an orientation consistent with the expected reaction path, with e.g.…”

Probing the Role of Active Site Water in the Sesquiterpene Cyclization Reaction Catalyzed by Aristolochene Synthase

“…18-21 Moreover, even in cyclases that do not generate alcohol products, trapped water molecules are occasionally observed in fully closed active sites complexed with analogues of the substrate, carbocation intermediates, or product. 22,23 Thus, a terpenoid cyclase must be able to control a trapped active site water molecule, when present, so that it reacts at the right time – or not at all – in the cyclization cascade.…”

“…25 However, recent high resolution crystal structures of ATAS complexes with the unreactive substrate analogue farnesyl- S -thiolodiphosphate (FSPP) as well as aza analogues of carbocation intermediates reveal trapped active site water molecules in each complex studied (Figure 1b). 23 Conserved water molecule "w" forms a hydrogen bond with N213 (a Mg 2+ B ligand in the NSE motif), N299, and S303; this water molecule is conserved in all crystal structures of ATAS in the closed conformation regardless of the ligand bound in the active site. 23 On the opposite side of the active site, conserved water molecules "w1" and "w2" are additionally trapped and hydrogen bonded with Q151 in the upper side of the active site, adjacent to the DDXXD motif.…”

“…23 Conserved water molecule "w" forms a hydrogen bond with N213 (a Mg 2+ B ligand in the NSE motif), N299, and S303; this water molecule is conserved in all crystal structures of ATAS in the closed conformation regardless of the ligand bound in the active site. 23 On the opposite side of the active site, conserved water molecules "w1" and "w2" are additionally trapped and hydrogen bonded with Q151 in the upper side of the active site, adjacent to the DDXXD motif. Finally, a Mg 2+ c -bound water molecule is exposed to the upper active site.…”