Epi-isozizaene synthase from Streptomyces coelicolor catalyzes the multistep cyclization of farnesyl diphosphate (2, FPP) to the tricyclic sesquiterpene hydrocarbon (+)-epi-isozizaene (3), which is converted in turn to the antibiotic albaflavenone (1) in a two-step, cytochrome P450-catalyzed oxidation. Competitive incubation of deuterated and nondeuterated samples of (3S)-NPP and (3RS)-NPP followed by GC-MS analysis of the degree of deuteration in the resulting labeled epi-isozizaene established that (3R)-NPP is the natural cyclization intermediate. Incubation of (3RS)-(Z)-[1-(2)H]NPP (4b) with epi-isozizaene synthase gave [11(anti)-(2)H]epi-isozizaene (3b), indicating that the S(N)' cyclization of 4 involves the predicted anti stereochemistry, consistent with the inference from earlier experiments with chirally deuterated FPP. Incubation of separate samples of [12,12,12-(2)H(3)]FPP (2d) and [13,13,13-(2)H(3)]FPP (2e) gave epi-isozizaenes 3d and 3e, thereby establishing the stereochemical course of the cyclization of the proposed intermediate acorenyl cation 6, as well as the stereochemistry of the successive 1,2-methyl migration and deprotonation that generate the final product. Further insights into the mechanism and the role of the enzyme came from site-directed mutagenesis of active site residues in two universally conserved Mg(2+)-binding domains and the identification of six minor sesquiterpene products 9-13 and 15 produced by the wild-type and mutant proteins. The aberrant products are believed to result from derailment and premature quenching of the normal intermediates of the cationic cyclization cascade.