Bioinformatics analyses enabled us to identify the hypothetical determinants of catalysis by CYP74 family enzymes. To examine their recognition, two mutant forms F295I and S297A of tomato allene oxide synthase LeAOS3 (CYP74C3) were prepared by site-directed mutagenesis. Both mutations dramatically altered the enzyme catalysis. Both mutant forms possessed the activity of hydroperoxide lyase, while the allene oxide synthase activity was either not detectable (F295I) or significantly reduced (S297A) compared to the wild-type LeAOS3. Thus, both sites 295 and 297 localized within the “I-helix central domain” (“oxygen binding domain”) are the primary determinants of CYP74 type of catalysis.

Keywords: Cytochrome P450; CYP74 family; Allene oxide synthase; Hydroperoxide lyase; Site-directed mutagenesis

Source: FEBS Letters (2008) Vol. 582, Issues 23-24, Pages 3423-3428