Judd, H. N. | Search bioRxiv

bioRxiv2025

Dynamic acetylation of a conserved lysine impacts glycerol kinase activity and abundance in the haloarchaeon Haloferax volcanii

Haloferax volcanii is a halophilic archaeon that preferentially utilizes glycerol as a carbon source, placing glycerol kinase (GK, glpK) at the center of its metabolism. In contrast to bacterial GKs, which are often regulated by allosteric inhibition, H. volcanii GK lacks this mode of control, indicating alternative regulatory mechanisms. Here, we show that lysine acetylation of H. volcanii GK enhances its activity and abundance during growth on glycerol, with K153 identified as the primary site of modification. Structural modeling and comparative genomics revealed that K153 resides in a conserved flexible loop common to haloarchaeal GKs. Carbon shifts from glucose to glycerol led to increased activity and enrichment of the K153-acetylated form, as determined by AQUA-MS. GK and the acetylation mimic K153Q supported growth on glycerol, while the non-acetylatable K153R variant did not. Thermal shift analysis showed that the K153R substitution reduced GK stability, while K153Q had no effect. Size exclusion chromatography indicated that GK is predominantly dimeric but forms a tetramer when purified from glycerol-grown cells and assayed with glycerol - coinciding with the highest K153 acetylation levels. Kinetic analysis revealed that K153 acetylation is required to maintain cooperative substrate binding, with the non-acetylatable K153R variant exhibiting a loss of allosteric behavior. The GNAT-family acetyltransferase Pat2 was found to acetylate GK at K153, and{Delta} pat2 mutants exhibited reduced GK protein abundance, linking Pat2 to regulation of GK. These results identify a dynamic, carbon source-responsive lysine acetylation mechanism that modulates GK, highlighting lysine acetylation as a key component of haloarchaeal metabolic regulation. IMPORTANCEPost-translational modifications allow microorganisms to rapidly adapt their metabolism to changing environmental conditions. Here, we uncover a carbon source-dependent acetylation mechanism that regulates GK activity and abundance in the halophilic archaeon H. volcanii. Unlike bacterial systems, where allosteric inhibitors control GK, H. volcanii relies on lysine acetylation to fine-tune and enhance enzymatic function based on nutrient availability. Our findings highlight acetylation at a conserved lysine as a key modulator of archaeal carbon metabolism, linking environmental signals directly to enzymatic activity and cellular fitness. This work expands our understanding of extremophile metabolic regulation and reveals how archaea deploy unique strategies to survive and thrive in environments that shift in carbon availability.

microbiology↗

bioRxiv2025

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. IMPORTANCEGNAT family homologs are widespread and diverse in their use of acyl-CoAs to acylate small molecules and proteins. These functions can be difficult to predict based on in silico analysis alone. Here we reveal a critical role for lysine acetylation in archaeal central carbon metabolism, identifying the GNAT family homolog Pat2 as an essential regulator of glycerol utilization in Haloferax volcanii. The findings expand our understanding of GNAT family acetyltransferases and highlight conserved mechanisms of metabolic control by PTM across domains of life.

microbiology↗