CYP52X1, representing a new cytochrome P450 subfamily, displays fatty acid hydroxylase activity and contributes to virulence and growth on insect cuticular substrates in the entomopathogenic fungus Beauveria bassiana

SHIZHU ZHANG; EMILIE WIDEMANN; GRAUSEM BERNARD; AGNES LESOT; FRANCK PINOT; NICOLÁS PEDRINI; NEMAT O. KEYHANI

JOURNAL OF BIOLOGICAL CHEMISTRY

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 2012 vol. 287 p. 13477 - 13486

0021-9258

Infection of insects by the entomopathogenic fungus Beauveria bassiana proceeds via attachment and penetration of the host cuticle. The outermost epicuticular layer or waxy layer of the insect represents a structure rich in hydrocarbons including abundant amounts of alkanes and fatty acids. A member of a novel cytochrome P450 subfamily, CYP52X1, implicated in hydrocarbon assimilation by B. bassiana was characterized.  B. bassiana targeted gene knockouts lacking Bbcyp52x1 displayed reduced virulence when topically applied to Galleria mellonella, but no reduction in virulence was noted when the insect cuticle was bypassed using an intrahemoceol injection assay. No significant growth defects were noted in the mutant as compared to the wild-type parent on any hydrocarbon substrates tested including alkanes and fatty acids. Insect epicuticle germination assays, however, showed reduced germination of DBbcyp52x1 conidia on grasshopper wings as compared to the wild-type parent. Complementation of the gene-knock with the full-length gene restored virulence and insect epicuticle germination to wild-type levels. Heterologous expression of CYP52X1 in yeast was used to characterize the substrate specificity of the enzyme. CYP52X1 displayed the highest activity against midrange fatty acids (C12:0 and C14:0) and epoxy stearic acid, 4-8 fold lower activity against C16:0, C18:1, and C18:2, and little to no activity against C9:0 and C18:0. Mass spec analysis of the product of the C16:0 reaction confirmed that the enzyme added a terminal hydroxyl to the substrate (w-hydroxylase). These data implicate CYP52X1 as contributing to the penetration of the host cuticle via facilitating the assimilation of insect epicuticle hydrocarbons.