Functional studies on the cytochrome P450 splice variants CYP4F3A and CYP4F3B unveil the basis for their distinct physiological functions.

The cytochrome P450 (P450) 4F family (CYP4F) are fatty acid ⍵-hydroxylases that catalyze the insertion of a hydroxyl group at the terminal carbon. The enzymes CYP4F3A and CYP4F3B are special cases among all other human P450 enzymes because they are derived from the same gene. The CYP4F3 gene undergoes alternative splicing, resulting in the 2 distinct enzymes. CYP4F3A is exclusively expressed in monocytes and deactivates leukotriene B4 as part of the anti-inflammatory response. Conversely, CYP4F3B is expressed in the liver and kidney where its major function is the production of the potent lipid mediator 20-hydroxyeicosatetraenoic acid from arachidonic acid. Despite these differences, they share a 93% amino acid sequence identity because of their shared gene locus. Both CYP4F3A and CYP4F3B are potential therapeutic targets for autoimmune disorders, cardiovascular diseases, and cancer. Because there is a significant gap in understanding enzyme function, their use as therapeutic targets has not been realized yet. To our knowledge, we present the first protocol for the generation of functional recombinant CYP4F3A and CYP4F3B to high purity. Catalytic assays with arachidonic acid and leukotriene B4 reveal a distinct substrate preference of both enzymes, which confirm their distinct body functions. Spectral analysis confirmed a different binding mode of arachidonic acid to the splice variants with a differential interaction with the respective active site. In addition, we tested the inhibitory effect of the CYP4 pan inhibitor HET0016 on both variants. In conclusion, we successfully implemented a robust protocol for the production of recombinant CYP4F3A and CYP4F3B, which paves the way for more in-depth mechanistic and structural studies and future directed drug design. SIGNIFICANCE STATEMENT: The splice variants CYP4F3A and CYP4F3B originate from the same gene but assume different functions in the human body. However, in-depth structural and functional studies are missing owing to the lack of robust protein expression protocols. In this study, we achieved the first generation of recombinant enzyme and conducted functional studies with fatty acid substrates and drugs, paving a way to a deeper understanding of these fascinating enzymes.