Role of the conserved distal heme asparagine in coral allene oxide synthase and human catalase

Abstract

Allene oxide synthase (AOS) metabolizes a specific fatty acid hydroperoxide to an unstable allene epoxide, a biosynthetic precursor of cyclopentanones such as jasmonic acid and marine clavulones. Plant AOS is a cytochrome P450 (CYP74A), whereas coral AOS (cAOS) is structurally related to catalase. The striking similarity of catalase and cAOS revealed by the X‐ray structures opens new questions on the structure‐function of both enzymes. Here we addressed the role of the conserved distal heme Asn in H2O2 breakdown in catalase and in metabolism of the natural hydroperoxide substrate 8R‐HPETE by cAOS. Point mutations were introduced by site directed mutagenesis and the proteins expressed in E. coli. Activities were assayed by UV spectrophotometry and cAOS products were identified by HPLC‐UV. In human catalase, the point mutations N148A, N148S, or N148D reduced Vmax and Km, with little net effect on kcat/Km. In cAOS, the mutations N137A, N137Q, N137S, N137D, and N137H drastically reduced the rate of reaction (to 0.8–4%), yet the mutants all formed the allene oxide as product. This is remarkable because there are many potential heme‐catalyzed transformations of fatty acid hydroperoxides. The distal heme Asn is not essential in either catalase or cAOS. Its conservation throughout evolution may relate to a role in enzyme stabilization and fine‐tuning of catalysis.Supported by NIH grant GM‐074888