Chrysanthemyl Diphosphate Synthase. The Relationship among Chain Elongation, Branching, and Cyclopropanation Reactions in the Isoprenoid Biosynthetic Pathway

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Abstract
The genes for chrysanthemyl diphosphate (CPP) synthase and farnesyl diphosphate (FPP) synthase from sagebrush, Artemisia tridentata spiciformis, were used to prepare a series of chimeric proteins to investigate the 1‘−4 chain elongation, 1‘−2 branching, and c1‘−2−3 cyclopropanation reactions that join isoprenoid units to build more complex structures. The two genes were modified by site-directed mutagenesis to generate an identical set of six unique restriction sites at identical locations. The locations were selected to place a restriction site between each of the five conserved regions found in prenyltransferases that catalyze chain elongation. A series of chimeric proteins were generated by replacing amino acids in FPP synthase, beginning at the N-terminus of the enzyme, with increasing stretches of peptide from CPP synthase. An analysis of the products produced by the chimeras revealed a transition from 1‘−4 chain elongation, to 1‘−2 branching, and ultimately to c1‘−2−3 cyclopropanation. These results demonstrate that the catalytic site for chain elongation, with minor modifications in its architecture, also catalyzes 1‘−2 branching and c1‘−2−3 cyclopropanation, and suggest that the branching and cyclopropanation reactions, in analogy to chain elongation, are electrophilic alkylations.