A Mechanistic Study of Isopenicillin N Formation Using Density Functional Theory

The catalytic mechanism of isopenicillin N formation by isopenicillin N synthase has been investigated using the density functional theory method B3LYP. The catalytic cycle is suggested to occur in 12 steps, where the closure of the four-membered β-lactam ring precedes the closure of the five-membered thiazolidine ring. Two of the reaction steps were found to have significant barriers of similar size, in line with experimental suggestions that these steps are rate-determining. One of these steps is the hydrogen atom transfer from the cysteinyl β-carbon, leading to formation of an oxo-ferryl complex and, in a subsequent step, to formation of the monocyclic β-lactam ring. The other significant barrier is obtained for the hydrogen atom transfer from the β-carbon of the valinyl group to the oxo-ligand of IPNS, initiating the formation of the thiazolidine ring. The last step of the catalytic reaction, which completes the closure of the thiazolidine ring by a bond formation between the Val-β-carbon and the Cys-sulfur, was found to be very exothermic. An important aspect obtained from the calculations is that the dioxygen ligand needs to be protonated prior to the Cys-β-C-H activation. Otherwise, this step would not be partly rate-limiting and would thus be in conflict with isotope experiments.