The
diterpene cyclase
taxadiene synthase from yew (Taxus) species transforms
geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committed step in the biosynthesis of the anti-
cancer drug Taxol.
Taxadiene synthase is translated as a preprotein bearing an N-terminal targeting sequence for localization to and processing in the plastids. Overexpression of the full-length preprotein in Escherichia coli and purification are compromised by host codon usage, inclusion body formation, and association with host chaperones, and the preprotein is catalytically impaired. Since the transit
peptide-mature
enzyme cleavage site could not be determined directly, a series of N-terminally truncated
enzymes was created by expression of the corresponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional
protein; however, deletion of 93 or more
amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. The pseudomature form of
taxadiene synthase having 60
amino acids deleted from the preprotein was found to be superior with respect to level of expression, ease of purification, solubility, stability, and catalytic activity with kinetics comparable to the native
enzyme. In addition to the major product, taxa-4(5),11(12)-diene (94%), this
enzyme produces a small amount of the isomeric taxa-4(20), 11(12)-diene ( approximately 5%), and a product tentatively identified as verticillene ( approximately 1%). Isotopically sensitive branching experiments utilizing (4R)-[4-(2)H(1)]
geranylgeranyl diphosphate confirmed that the two
taxadiene isomers, and a third (taxa-3(4),11(12)-diene), are derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the
enzyme to utilize 2, 7-cyclogeranylgeranyl
diphosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the
diphosphate ester and macrocyclization to the verticillyl intermediate, followed by a secondary cyclization to the taxenyl
cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring closure). Two potential mechanism-based inhibitors were tested with recombinant
taxadiene synthase but neither provided time-dependent inactivation nor afforded more than modest competitive inhibition.