Protein homology modeling‐informed active site characterization of a bacterial prostaglandin synthase ortholog from Nitrosomonas europaea

Abstract

An open reading frame in the genome of Nitrosomonas europaea has been annotated as a prostaglandin H synthase (PGHS). We have expressed the protein, which demonstrates peroxidase activity with both hydrogen peroxide and lipid hydroperoxide substrates. Dioxygenase activity was not observed. In this study, we generate models of the N. europaea peroxidase to understand the structural explanation for its observed function. Using SWISS-MODEL, protein homology models were generated against PGHS, peroxidase, and dioxygenase templates. Using AutoDock Vina, the substrate 13(S)-hydroperoxy-(9Z, 11E)-octadecadienoic acid was inserted into the active site of each model to identify potential amino acids critical for activity. A conserved di-tyrosyl structure was observed in several unique models generated against redox enzyme templates. Amino acids in the active site predicted in the protein homology models were modified by site-directed mutagenesis. Specifically, the conserved tyrosyl structure was mutated to elucidate the importance of the structure in addition to the individual residues. Wild-type and mutant enzymes were expressed in BL21 cells and purified by Ni-NTA and Sephacryl S-200 chromatography. Kinetic assays using hydrogen peroxide and lipid hydroperoxide substrates are in progress to compare the mutant and wild-type enzyme activity. Identifying critical residues will contribute to our understanding of the evolutionary development of mammalian prostaglandin synthase.