Characterization Of Human CYP4Z1 By Mutation Analysis

Cytochrome P450 enzymes(CYPs or P450s)are a large superfamily of hemoproteins present in all biological kingdoms that are best known for catalyzing hydroxylation reactions.In humans,there are 57 functional CYP enzymes that oxidize endogenous substances and exogenous substances including drugs and environmental compounds.In this study is focused on human CYP4Z1,which is an “orphan” CYP that belongs to the CYP4 family.CYP4Z1 remains an understudied enzyme despite its association with poor prognosis and overexpression in breast cancer.Breast cancer is a malignant tumor that occurs in the epithelial tissue of the breast,and it is also the most commonly diagnosed cancer among women with almost 2.1 million newly diagnosed in 2018 all over the world,as well as the leading cause of cancer deaths in women which accounts for 15 % of all cancer deaths.Therefore,research on breast cancer treatment is particularly important and necessary.Because of its strong overexpression in breast cancer cells,CYP4Z1 has been suggested as a target for a prodrug-based breast cancer therapy.Ideally,such a prodrug is converted into an anti-cancerous compound in a reaction that can only be catalyzed by CYP4Z1.In order to conduct the prodrug research and design a suited CYP4Z1-based prodrug,we need to understand the structure-activity relationships of CYP4Z1,which was therefore the main goal of this study.Our group was the first to report catalytic activities of CYP4Z1 after recombinant expression of the enzyme in the fission yeast Schizosaccharomyces pombe.But since CYP4Z1 is a membrane-bound protein,there is still no experimentally solved structure of this enzyme.Our cooperation partner Prof.Gerhard Wolber(Free University Berlin,Germany)had previously started to rationalize the enzyme’s properties by creating a homology model and performing substrate docking.It was the aim of the present study to conduct extended mutation analysis of this enzyme in order to improve understanding of the substrate binding mode and to allow for fine-tuning of the previously established homology model.Activity assays were done using permeabilized recombinant fission yeast cells(“enzyme bags”),which can efficiently be employed in a similar way as microsomal preparations but are much easier to fabricate.In a combined in vitro and in silico approach,we show for the first time that residue Arg487 plays an important role in substrate recognition and binding of CYP4Z1.Using a large array of recombinant CYP4Z1 mutants we show that,apart from Asn381,the other postulated binding residues(such as Ser113,Ser222,and Ser383)only play an auxiliary role in substrate recognition and binding.Different substrate interaction motifs were identified via dynamic pharmacophores(dynophores)and their impact on catalytically competent substrate binding was classified.These new insights on the substrate recognition and binding mode represent an important step towards the rational design of CYP4Z1 prodrugs and guide further investigations into the so far poorly understood physiological role of CYP4Z1.