Couto-Rodriguez, R. L. | Search bioRxiv

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↗

bioRxiv2022

TrmB family transcription factor as a thiol-based regulator of oxidative stress response

Oxidative stress causes cellular damage including DNA mutations, protein dysfunction and loss of membrane integrity. Here we discovered TrmB (transcription regulator of mal operon) family proteins (Pfam PF01978) composed of a single winged-helix DNA binding domain (InterPro IPR002831) can function as thiol-based transcriptional regulators of oxidative stress responses. Using the archaeon Haloferax volcanii as a model system, we demonstrate that the TrmB-like OxsR is important for recovery of cells from hypochlorite stress. OxsR is shown to bind specific regions of genomic DNA, particularly during hypochlorite stress. OxsR-bound intergenic regions were found proximal to oxidative stress operons including genes associated with thiol relay and low molecular weight thiol biosynthesis. Further analysis of a subset of these sites, revealed OxsR to function during hypochlorite stress as a transcriptional activator and repressor. OxsR was shown to require a conserved cysteine (C24) for function and to use a CG-rich motif upstream of conserved BRE/TATA box promoter elements for transcriptional activation. Protein modeling suggested the C24 is located at a homodimer interface formed by antiparallel helices, and that oxidation of this cysteine would result in the formation of an intersubunit disulfide bond. This covalent linkage may promote stabilization of an OxsR homodimer with the enhanced DNA binding properties observed in the presence of hypochlorite stress. The phylogenetic distribution TrmB family proteins, like OxsR, that have a single winged-helix DNA binding domain and conserved cysteine residue suggests this type of redox signaling mechanism is widespread in Archaea. ImportanceTrmB-like proteins, while not yet correlated with redox stress, are found in bacteria and widespread in archaea. Here we expand annotation of a large group of TrmB-like single winged-helix DNA binding domain proteins from diverse archaea to function as thiol-based transcriptional regulators of oxidative stress response. Using Haloferax volcanii as a model, we reveal the TrmB-like OxsR functions during hypochlorite stress as a transcriptional activator and repressor of an extensive gene co-expression network associated with thiol relay and other related activities. A conserved cysteine residue of OxxR serves as the thiol-based sensor for this function and likely forms an intersubunit disulfide bond during hypochlorite stress that stabilizes a homodimeric configuration with enhanced DNA binding properties. A CG-rich DNA motif in the promoter region of a subset of sites identified to be OxsR-bound is required for regulation; however, not all sites have this motif suggesting added complexity to the regulatory network.

microbiology↗