GNAT family Pat2 lysine acetylation of glycerol kinase and its key role in glycerol metabolism in hypersaline-adapted archaea

Lysine acetylation is a widespread post-translational modification (PTM) involved in regulating key biological processes including central metabolism and chromatin dynamics, yet its roles in archaea remain poorly understood. Here, we investigated two GNAT (Gcn5-related N-acetyltransferase) family homologs, pat1 and pat2 , in the halophilic archaeon Haloferax volcanii (Hv). We found that a pat2 mutant exhibited impaired growth and premature cell death on glycerol, a phenotype not observed in the parent strain, pat1 mutant, or during growth on glucose. Complementation with plasmid-expressed pat2 restored growth on glycerol, confirming this biological role. In vitro assays demonstrated that HvPat2 catalyzes the lysine acetylation of HvGlpK, a glycerol kinase essential for glycerol metabolism. Computational modeling predicted that HvPat2 residues E105, Y154, V110, and N147 may form hydrogen bonds with acetyl-CoA. To assess the functional importance of these residues, alanine substitutions were introduced at each site. Growth assays revealed that E105A and Y154A variants failed to restore growth on glycerol, while V110A and N147A had no significant effect. In vitro , HvPat2 Y154A, E105A, and V110A lacked acetyltransferase activity toward GlpK, whereas N147A retained partial activity. HvPat2 Y154A co-purified with a protein partner, potentially explaining the discrepancy between in vivo and in vitro results. These findings highlight the critical role of the GNAT HvPat2 in mediating lysine acetylation in regulating glycerol metabolism in archaea and offer mechanistic insight into GNAT family acetyltransferases.