Bornyl diphosphate synthase: Structure and strategy for carbocation manipulation by a terpenoid cyclase

Abstract

The x-ray crystal structure of dimeric (؉)-bornyl diphosphate synthase, a metal-requiring monoterpene cyclase from Salvia officinalis, is reported at 2.0-Å resolution. Each monomer contains two ␣-helical domains: the C-terminal domain catalyzes the cyclization of geranyl diphosphate, orienting and stabilizing multiple reactive carbocation intermediates; the N-terminal domain has no clearly defined function, although its N terminus caps the active site in the C-terminal domain during catalysis. Structures of complexes with aza analogues of substrate and carbocation intermediates, as well as complexes with pyrophosphate and bornyl diphosphate, provide ''snapshots'' of the terpene cyclization cascade.

16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: Potent Mechanism-Based Inhibitors of Recombinant ent-Kaurene Synthase from Arabidopsis thaliana

“…Similar findings have been reported with other terpene synthases, 17,18 suggesting that the use of the charged PP·Mg complex as a counter ion to stabilize proximal secondary carbocation intermediates may be a relatively common enzymatic mechanism utilized by these enzymes. However, such counter ion stabilization seems to be limited to carbocations located relatively close to the original PP position, as indicated by the weaker synergy exhibited by distal aza analogues and structural studies.…”

“…However, such counter ion stabilization seems to be limited to carbocations located relatively close to the original PP position, as indicated by the weaker synergy exhibited by distal aza analogues and structural studies. 17,19 Indeed, it has been suggested that the PP·Mg anion may drive reactions towards localization of the carbocation intermediates close to the negative charge, particularly in the absence of other stabilizing influences in the active site. 11 Finally, the fact that ent-beyeran-16-yl PP does not appear to be a free intermediate further emphasizes the tight control KS must exert over the ionized carbocation intermediates and PP·Mg anion within its active site to prevent recombination of the closely associated ion pair.…”

“…However, the protonated forms of 2-azabornane and the isomeric 15-azapimarenes are notable as effective mimics for high energy, pyramidalized secondary born-2-yl and pimaren-15-yl carbocation/PP·Mg ion pairs presumably generated at the active sites of bornyl diphosphate and abietadiene synthases. 17,18 Azabornane and other aza-terpenes have served as useful active site probes in X-ray crystallography of mono-and sesquiterpene cyclases. 19 In this paper we report the synthesis and characterization of 16-aza-ent-beyerane and 16-aza-ent-trachylobane (12 and 13, Fig 2), as well as the potential intermediate entbeyeran-16-yl PP (11), and a preliminary kinetic evaluation of the compounds as mechanism-based inhibitors of recombinant KS from Arabidopsis thaliana.…”

“…21 Cleavage of the D ring of ent-beyeranone was accomplished by Beckmann fragmentation of the oxime derivative (17). The oxime was formed as a single isomer in high yield (NH 2 OH•HCl, pyr) and presumed to have the less sterically crowded E configuration.…”

Nature as organic chemist

“…These first structures were followed shortly thereafter by those of trichodiene synthase 79 and aristolochene synthase (Figure 11b), 80,81 In collaboration with Rod Croteau, David's laboratory also solved the crystal structure of the monoterpene synthase, bornyl diphosphate synthase. 82 These studies confirmed the universal all-helical fold of the sesquiterpene and monoterpene synthases, and have provided detailed pictures of the organization of the active sites of these remarkable, synthetically versatile cyclases. 76,[83][84][85][86] Complementing and enriching these studies, Prof Dean J Tantillo (U. C. Davis) has deepened the understanding of the mechanisms and energetics of terpene cyclization reactions through detailed quantum chemical calculations of the reaction pathways linking carbocationic intermediates and transition state structures for a wide variety of terpene synthase-catalyzed reactions.…”