Isochorismate Pyruvate Lyase: A Pericyclic Reaction Mechanism?

Abstract

Isochorismate pyruvate lyase (IPL) catalyzes the cleavage of isochorismate to give salicylate and pyruvate, a key step in bacterial siderophore biosynthesis. We investigated the enzyme from Pseudomonas aeruginosa using isochorismate selectively deuterated at C2 as a substrate. Monitoring the reaction by ²H NMR spectroscopy revealed that the label is quantitatively transferred from C2 to C9, producing stoichiometric amounts of [3-²H]pyruvate as product. Moreover, the deuterium kinetic isotope effect of 2.34 ± 0.08 on k_cat indicates that C−H cleavage is significantly rate limiting. Consistent with these data, hybrid density functional theory (HDFT) calculations at the Becke3LYP/DZ+(2d,p) level of theory predict a concerted but highly asynchronous pericyclic transition structure, in which carbon−oxygen bond cleavage is more advanced than hydrogen atom transfer from C2 to C9; the calculated ²H isotope effect of 2.22 at C2 is in excellent accord with the experimental value. Together, these findings indicate that IPL should be added to the small set of proteins that are known to catalyze pericyclic reactions. They also raise the possibility that enzymes, such as chorismate pyruvate lyase, salicylate synthase, 4-amino-4-deoxychorismate lyase, and anthranilate synthase, which accelerate formally similar reaction steps, may also exploit pericyclic mechanisms.