ZDHHC13 Is a Catalytically Deficient Protein S -Acyltransferase That Functions via a Noncanonical Mechanism

S-Acylation is the addition of fatty acids to cysteine residues to regulate protein function and localization. S-Acylation is catalyzed by ZDHHC (Asp-His-His-Cys) protein S-acyltransferases (PATs), which S-acylate protein substrates by first auto-S-acylating the catalytic cysteine of the DHHC active site followed by transfer to the substrate. ZDHHC13 and ZDHHC17 are related ankyrin repeat domain (ANK) PATs that S-acylate neuronal proteins, including Huntingtin (HTT), the protein mutated in Huntington disease. However, unlike ZDHHC17 and other human PATs, ZDHHC13 possesses a noncanonical DQHC active site. As the first histidine is essential for auto-S-acylation, it is unclear if ZDHHC13 is catalytically active. Our phylogenetic analysis of eukaryotic ANK PATs showed that while the DHHC of ZDHHC17 orthologues is highly conserved, the motif is variable among ZDHHC13 orthologues, suggesting evolution independent of catalytic activity. We found that the ZDHHC13 catalytic cysteine is indeed S-acylated in cells, albeit substantially less than ZDHHC17 or ZDHHC20. We also confirmed minimal autoacylation activity in vitro with purified ZDHHC13. While wild-type (WT) ZDHHC13 increased S-acylation of an HTT1-588 fragment in cells, surprisingly, catalytically dead DQHS ZDHHC13 facilitated HTT1-588 S-acylation equally. This suggests that the ZDHHC13 catalytic cysteine is not required for substrate S-acylation and instead ZDHHC13 contributes to S-acylation via an indirect mechanism. Furthermore, ZDHHC13 was identified in a high-molecular-weight complex, suggesting a scaffolding or accessory role in S-acylation. This work broadens our understanding of this noncanonical PAT and lays the foundation for future mechanistic and structural studies.