Heterologous expression and characterization of a "pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction

The diterpene cyclase taxadiene synthase from yew (Taxus) species transform s geranylgeranyl diphosphate to taxa-4(5),11(12)-diene as the first committ ed step in the biosynthesis of the anti-cancer drug Taxol. Taxadiene syntha se is translated as a preprotein bearing an N-terminal targeting sequence f or localization to and processing in the plastids. Overexpression of the fu ll-length preprotein in Escherichia coli and purification are compromised b y host codon usage, inclusion body formation, and association with host cha perones, and the preprotein is catalytically impaired. Since the transit pe ptide-mature enzyme cleavage site could not be determined directly, a serie s of N-terminally truncated enzymes was created by expression of the corres ponding cDNAs from a suitable vector, and each was purified and kinetically evaluated. Deletion of up to 79 residues yielded functional protein; howev er, deletion of 93 or more amino acids resulted in complete elimination of activity, implying a structural or catalytic role for the amino terminus. T he pseudomature form of taxadiene synthase having 60 amino acids deleted fr om the preprotein was found to be superior with respect to level of express ion, ease of purification, solubility, stability, and catalytic activity wi th kinetics comparable to the native enzyme. In addition to the major produ ct, taxa-4(5),11(12)-diene (94%), this enzyme produces a small amount of th e isomeric taxa-4(20),11(12)-diene (similar to 5%), and a product tentative ly identified as verticillene (similar to 1%). Isotopically sensitive branc hing experiments utilizing (4R)-[4-2H(1)]geranylgeranyl diphosphate confirm ed that the two taxadiene isomers, and a third (taxa-3(4),11(12)-diene), ar e derived from the same intermediate taxenyl C4-carbocation. These results, along with the failure of the enzyme to utilize 2,7-cyclogeranylgeranyl di phosphate as an alternate substrate, indicate that the reaction proceeds by initial ionization of the diphosphate ester and macrocyclization to the ve rticillyl intermediate, followed by a secondary cyclization to the taxenyl cation and deprotonation (i.e., formation of the A-ring prior to B/C-ring c losure). Two potential mechanism-based inhibitors were tested with recombin ant taxadiene synthase but neither provided time dependent inactivation nor afforded more than modest competitive inhibition. (C) 2000 Academic Press.