The Mechanism for Isopenicillin N Synthase from Density-Functional Modeling Highlights the Similarities with Other Enzymes in the 2-His-1-carboxylate Family<sup>†</sup> Marcus Lundberg Per E. M. Siegbahn Keiji Morokuma 10.1021/bi701577q.s036 https://acs.figshare.com/articles/dataset/The_Mechanism_for_Isopenicillin_N_Synthase_from_Density_Functional_Modeling_Highlights_the_Similarities_with_Other_Enzymes_in_the_2_His_1_carboxylate_Family_sup_sup_/2961778 Isopenicillin N synthase (IPNS) catalyzes a key step in the biosynthesis of the important β-lactam antibiotics penicillins and cephalosporins. Density-functional calculations with the B3LYP functional are used to propose a detailed mechanism for this reaction. The results support the general scheme outlined from experimental observations, with formation of a four-membered β-lactam ring followed by formation of a five-membered thiazolidine ring. However, an alternative mechanism for the heterolytic O−O bond cleavage and β-lactam ring formation steps is proposed. The former part involves protonation of the distal oxygen by an iron-bound water ligand. This mechanism highlights the strong similarities that exist between IPNS and other enzymes of the 2-histidine-1-carboxylate family, especially pterin-dependent amino acid hydroxylases and α-keto acid-dependent dioxygenases. Both activation of the cysteine β-C−H bond by an iron-bound superoxo radical and activation of the valine β-C−H bond by a ferryl−oxo species show reaction barriers close to the experimentally measured one. These results are in agreement with kinetic isotope experiments that suggest both C−H bond activation steps to be partially rate limiting. The ring formation sequence is determined by the relative strengths of the two C−H bonds. Only the ferryl−oxo intermediate is capable of activating the stronger valine β-C−H bond. 2008-01-22 00:00:00 Other Enzymes results support activation bond isotope experiments alternative mechanism lactam IPNS ring formation sequence acid hydroxylases Isopenicillin N Synthase B 3LYP