The self-sufficient Aspergillus fumigatus Δ9-fatty acid desaturase SdeA requires a fused cytochrome b5 domain and hinge-dependent self-association for virulence

Fungal Δ9-fatty acid desaturases are self-sufficient enzymes in which the electron-donating cytochrome b5 (Cytb5) module is fused to the catalytic core, yet whether this covalent linkage is architecturally required has remained untested. Here we show that in the human pathogen Aspergillus fumigatus the Cytb5 module of the Δ9-desaturase SdeA must act in cis, within a self-associated complex organized by a hinge segment. AlphaFold3 modeling predicts a dimer in which the Cytb5 domain of one protomer approaches the other's di-iron center at a distance compatible with electron tunneling, and reciprocal co-immunoprecipitation shows that full-length SdeA self-associates in vivo. The recombinant Cytb5 module displays the spectral signatures of a genuine heme-binding redox domain. Nevertheless, deleting the Cytb5 domain, substituting its heme-coordinating histidines, or removing the 25-residue hinge each abolishes growth and virulence in Galleria mellonella without disrupting endoplasmic reticulum targeting, and a detached Cytb5-containing segment does not restore function. Hinge deletion abolishes stable SdeA self-association, identifying this segment as a structural organizer of the catalytically competent complex. Finally, we find no evidence that the sdeB locus provides a backup Δ9-desaturase. These results define the architectural requirements of a self-sufficient fungal desaturase and identify vulnerabilities absent from the human Δ9-desaturase for antifungal targeting.