Mechanism of Abietadiene Synthase Catalysis:  Stereochemistry and Stabilization of the Cryptic Pimarenyl Carbocation Intermediates

Abstract

Abietadiene synthase (AS) catalyzes the complex cyclization-rearrangement of (E,E,E)-geranylgeranyl diphosphate (8, GGPP) to a mixture of abietadiene (1a), double bond isomers 2a-4a and pimaradienes 5a-7a as a key step in the biosynthesis of the abietane resin acid constituents (1b-4b) of conifer oleoresin. The reaction proceeds at two active sites by way of the intermediate, copalyl diphosphate (9). In the second site, a putative tricyclic pimaradiene or pimarenyl(+) carbocation intermediate of undefined C13 stereochemistry and annular double bond position is formed. Three 8-oxy-17-nor analogues of 9 (17 and 19a,b) and three isomeric 15,16-bisnorpimarenyl-N-methylamines (26a-c) were synthesized and evaluated as alternative substrates and/or inhibitors for recombinant AS from grand fir. The stereospecific cyclization of 8 alpha-hydroxy-17-nor CPP (19a) to 17-normanoyl oxide (20a) and the higher inhibitory potency of the norpimarenylamine 26a (K(i) = 0.1 nM) both suggest pimarenyl intermediates having the 13 beta methyl configuration and 8,14-double bond corresponding to sandaracopimaradiene (5a). The 2000-fold stimulation of inhibition by 26a in the presence of inorganic pyrophosphate indicates an important role for carbocation/OPP anion stabilization of the secondary sandaracopimaren-15-yl(+) ion. The failure of 8 beta-hydroxy-17-nor CPP (19b) to undergo enzymatic cyclization was taken as evidence that 9 is bound with a "coplanar" side chain conformation and that the S(N)' cyclization occurs on the 17 alpha face. The routing of the sandarcopimara-15-en-8-yl carbocation toward various diterpenes in biogenetic schemes is attributed to differing conformations of ring C and/or orientations of the C13 vinyl group in the active sites of the corresponding diterpene cyclases.

Functional Characterization of Nine Norway Spruce TPS Genes and Evolution of Gymnosperm Terpene Synthases of the TPS-d Subfamily

“…Recent work on abietadiene synthase (AgAS) from grand fir (Ravn et al, , 2002Peters et al, 2000Peters et al, , 2001Peters et al, , 2003 has dissected the mechanism of this bifunctional conifer di-TPS. AgAS is very similar to Norway spruce PaTPS-LAS and PaTPS-Iso, thereby making the recent findings relevant to the mechanisms of these two enzymes as well.…”

Functional plasticity of paralogous diterpene synthases involved in conifer defense

“…To date, only a handful of di-TPSs involved in diterpene resin acid biosynthesis have been characterized, with all of them originating from gymnosperm trees (2). A series of papers from Croteau, Peters, and coworkers (14,15,(19)(20)(21)(22)(23) studied an abietadiene synthase (AS) from Grand fir [Abies grandis (Ag)] to establish the general mechanism and specific features of this type of di-TPS. Two closely related di-TPSs identified in Norway spruce [Picea abies (Pa)] have 91% amino acid identity but nonoverlapping product profiles (5).…”

“…1]. The (ϩ)-CPP then freely diffuses to a second active site (class I) in the C-terminal domain, containing the Mg 2ϩ -binding DDXXD motif that catalyzes the diphosphate ionization-initiated cyclization of (ϩ)-CPP to produce the C8-sandaracopimarenyl cation intermediate (21)(22)(23) (Fig. 1, routes a and c).…”

A Single Residue Switch Converts Abietadiene Synthase into a Pimaradiene Specific Cyclase

“…7 Later mechanistic work with recombinant AgAS established the intermediacy of isopimarenyl + (5) intermediates in enzymatic production of abietadienes from copalyl diphosphate (CPP, 6). 4 In addition, AgAS has been shown to produce sandaracopimara-8(14),15-diene (7) as a minor component (∼2%) of its product output, 8 suggesting 7 as a potential stable intermediate. However, labeling studies demonstrated intramolecular proton transfer, 9 and 7 is not converted to abietadienes by AgAS, 2 indicating that if 7 is a true intermediate it is only transiently formed en route to abietadienes in the AgAS active site.…”

“…4,13 In particular, the secondary carbocations are significantly closer to the pyrophosphate than the preceding tertiary carbocations (e.g., Scheme 1). Accordingly, we hypothesize that the introduced hydroxyl group, at the relevant position in either AgAS or the previously examined ent-kaurene synthases, simply acts to stabilize pimar-15-en-8-yl + intermediates long enough for deprotonation to occur.…”