Mechanism of abietadiene synthase catalysis: stereochemistry and stabilization of the cryptic pimarenyl carbocation intermediates.
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.
Ravn
Matthew M
MM
Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, Illinois 61801, USA.
Peters
Reuben J
RJ
Coates
Robert M
RM
Croteau
Rodney
R
D002384:Catalysis; D004791:Enzyme Inhibitors / Q000138:chemical synthesis / Q000737:chemistry; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D018716:Molecular Mimicry; D010755:Organophosphates / Q000138:chemical synthesis / Q000737:chemistry*; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism
D004791:Enzyme Inhibitors; D010755:Organophosphates; D010936:Plant Extracts; C408747:copalyl diphosphate; C018620:oleoresins; D007535:Isomerases; C088741:abietadiene cyclase