Fig. 5.

(A) Formation of presegetalin A1 [20–32] was observed under single turnover conditions by LC-MS. After a 5-min incubation, wild-type PCY1 was able to catalyze formation of the acyl-enzyme intermediate, producing the presegetalin A1 [20–32] by-product. The H695A mutant did not catalyze formation of presegetalin A1 [20–32], indicating that it could not form the acyl-enzyme intermediate. (B) An 18-h incubation of H695A with 20 mM imidazole allowed for the production of both linear [GVPVWA] and cyclic segetalin A. However, the H695A variant produces a significantly greater amount of hydrolytic, linear product (ratio of cyclic:linear product of ∼3.7:1), whereas wild-type PCY1 almost exclusively produces the macrocyclic product (Fig. 2).