(-)-
Abietic acid, the principal
diterpenoid resin
acid of the
wound-induced oleoresin secreted by grand fir (Abies grandis), is synthesized by the cyclization of
geranylgeranyl diphosphate to (-)-abieta-7(8),13(14)-diene, followed by sequential three-step oxidation of the C-18 methyl group of the
olefin to a carboxyl function. The
enzyme catalyzing the cyclization reaction,
abietadiene synthase, was purified from stems of wounded grand fir saplings and was digested with
trypsin. Amino acid sequence information from the resulting
peptides allowed construction of degenerate
oligonucleotide primers, which amplified a 551-base pair fragment from a
wound-induced stem cDNA library. This hybridization probe was then utilized to screen the
wound-induced stem cDNA library, from which three
cDNA clones were isolated that were functionally expressed in Escherichia coli, thereby confirming that a single
protein catalyzes the complex, multistep cyclization of
geranylgeranyl diphosphate to
abietadiene.
cDNA isolate Ac22.1, which yielded the highest expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino
acid open reading frame that included a putative plastidial transit
peptide. Deduced amino acid sequence comparison to other
terpene cyclases revealed an amino-terminal region of the
abietadiene synthase, which resembles those of
enzymes that employ substrate double bond protonation to initiate the carbocationic reaction cascade, and a carboxyl-terminal region of the synthase, which resembles those of
enzymes that employ ionization of the substrate allylic
diphosphate ester function to initiate the cyclization reaction. This apparent fusion of segments of the two distinct
terpenoid cyclase types is consistent with the novel mechanism of the bifunctional
abietadiene synthase in catalyzing both protonation-initiated and ionization-initiated cyclization steps.