GNAT family Pat2 is required for long-term survival on glycerol and catalyzes lysine acetylation of glycerol kinase in hypersaline-adapted archaea

Lysine acetylation is a widespread post-translational modification that regulates key biological processes including metabolism and chromatin dynamics, yet its roles in archaea remain poorly understood. Here, we investigated two Gcn5-related N-acetyltransferase (GNAT) family homologs, <i>pat1</i> and <i>pat2</i>, in the halophilic archaeon <i>Haloferax volcanii</i> (Hv). A ∆<i>pat2</i> mutant exhibited premature cell death on glycerol, a phenotype not observed in the parent strain, ∆<i>pat1</i> mutant, or on glucose. Complementation of the ∆<i>pat2</i> mutant with plasmid-expressed <i>pat2</i> restored survival on glycerol, confirming the essential role of <i>pat2</i> in this process. <i>In vitro</i> assays revealed HvPat2 catalyzes lysine acetylation of glycerol kinase, HvGlpK, an enzyme required for growth on glycerol. Computational modeling predicted that HvPat2 residues E105, Y154, V110, and N147 may form hydrogen bonds with acetyl-CoA. To assess the importance of these residues, alanine substitutions were introduced. N147A and V110A complemented the ∆<i>pat2</i> mutant for survival on glycerol but showed little or no activity in acetylating HvGlpK <i>in vitro</i>. E105A and Y154A had the most pronounced effects, failing to restore ∆<i>pat2</i> survival on glycerol and showing no catalytic activity toward HvGlpK acetylation. Moreover, the Y154A variant co-purified with HVO_2384, a tandem CBS domain protein with a C-terminal ribosome hibernation factor domain, suggesting an additional regulatory interaction. These findings highlight the critical role of the GNAT HvPat2 in survival on glycerol, reveal its ability to acetylate a central glycerol metabolism enzyme in archaea, and offer mechanistic insight into GNAT family acetyltransferases.IMPORTANCEGNAT family homologs are widespread and diverse in their use of acyl-CoAs to acylate small molecules and proteins, functions difficult to predict based on <i>in silico</i> analysis alone. Here, we reveal a critical role for lysine acetylation in archaeal central carbon metabolism, identifying the GNAT family Pat2 of <i>Haloferax volcanii</i> as essential for long-term survival on glycerol (compared to glucose) and capable of mediating the lysine acetylation of glycerol kinase, a key enzyme in glycerol metabolism. Pat2 residues important for catalytic activity and a putative regulatory partner (HVO_2384) are also identified. The findings expand our understanding of GNAT family acyltransferases and highlight conserved mechanisms of metabolic control by post-translational modification across domains of life.