Structure-function studies of the non-heme iron active site of isopenicillin N synthase: some implications for catalysis

Isopenicillin N synthase (IPNS) is a non-heme ferrous iron-dependent oxygen ase that catalyzes the ring closure of delta-(L-alpha-aminoadipoyl)-L-cyste inyl-D-valine (ACV) to form isopenicillin N. Spectroscopic studies and the crystal structure of IPNS show that the iron atom in the active species is coordinated to two histidine and one aspartic acid residues, and to ACV, di oxygen and H2O. We previously showed by site-directed mutagenesis that resi dues His212, Asp214 and His268 in the IPNS of Streptomyces jumonjinensis ar e essential for activity and correspond to the iron ligands identified by c rystallography. To evaluate the importance of the nature of the protein lig ands for activity, His214 and His268 were exchanged with asparagine, aspart ic acid and glutamine, and Asp214 replaced with glutamic acid, histidine an d cysteine, each of which has the potential to bind iron. Only the Asp214Gl u mutant retained activity, similar to 1% that of the wild type. To determi ne the importance of the spatial arrangement of the protein ligands for act ivity, His212 and His268 were separately exchanged with Asp214; both mutant enzymes were completely defective. These findings establish that IPNS acti vity depends critically on the presence of two histidine and one carboxylat e ligands in a unique spatial arrangement within the active site. Molecular modeling studies of the active site employing the S. jumonjinensis IPNS cr ystal structure support this view. Measurements of iron binding by the wild type and the Asp214Glu, Asp214His and Asp214Cys-modified proteins suggest that Asp214 may have a role in catalysis as well as in iron coordination. ( C) 2000 Elsevier Science B.V. All rights reserved.