ABIETADIENE SYNTHASE FROM GRAND FIR (ABIES GRANDIS) - CDNA ISOLATION,CHARACTERIZATION, AND BACTERIAL EXPRESSION OF A BIFUNCTIONAL DITERPENE CYCLASE INVOLVED IN RESIN ACID BIOSYNTHESIS

(-)-Abietic acid, the principal diterpenoid resin acid of the wound-in duced 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 me thyl group of the olefin to a carboxyl function, The enzyme catalyzing the cyclization reaction, abietadiene synthase, was purified from ste ms of wounded grand fir saplings and was digested with trypsin, Amino acid sequence information from the resulting peptides allowed construc tion of degenerate oligonucleotide primers, which amplified a 551-base pair fragment from a wound-induced stem cDNA library, This hybridizat ion probe was then utilized to screen the wound-induced stern cDNA lib rary, from which three cDNA clones were isolated that were functionall y expressed in Escherichia coli, thereby confirming that a single prot ein catalyzes the complex, multistep cyclization of geranylgeranyl dip hosphate tea abietadiene, cDNA isolate Ac22.1, which yielded the highe st expressed level of cyclase activity, was 2861 base pairs in length and encoded an 868-amino acid open reading frame that included a putat ive plastidial transit peptide, Deduced amino acid sequence comparison to other terpene cyclases revealed an amino-terminal region of the ab ietadiene synthase, which resembles those of enzymes that employ subst rate double bond protonation to initiate the carbocationic reaction ca scade, and a carboxyl-terminal region of the synthase, which resembles those of enzymes that employ ionization of the substrate allylic diph osphate ester function to initiate the cyclization reaction. This appa rent fusion of segments of the two distinct terpenoid cyclase types is consistent with the novel mechanism of the bifunctional abietadiene s ynthase in catalyzing both protonation-initiated and ionization-initia ted cyclization steps.