Catalysis by allene oxide synthases (CYP74A and CYP74C): Alterations by the Phe/Leu mutation at the SRS-1 region

Abstract

The CYP74 family of cytochromes P450 includes four fatty acid hydroperoxide metabolizing enzymes: allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase, and epoxyalcohol synthase (EAS). All P450s have six substrate recognition sites (SRSs) in their structures. Some CYP74 mutations in SRSs leading to their interconversions including substitutions in “F/L toggle” (SRS-1 region) were reported before. For further elucidation of the role of this site in CYP74 catalysis, the effect of Phe/Leu mutation on the specificity of selected AOSs was studied in the present work. Mutant forms of ZmAOS1 (CYP74A19, Zea mays), LeAOS3 (CYP74C3, Lycopersicon esculentum), and PpAOS2 (CYP74A8, Physcomitrella patens) acquired partial EAS activity. Mutant forms of ZmAOS1 and PpAOS2 possessed additional HPL activities. The results validate the significance of the “F/L toggle” as a catalytic determinant of CYP74s, as well as the importance of the conserved Phe at this site for the AOS catalysis.