Sanchez Karol M, Addagarla Manasa, Judd Heather, Wang Xin, Maupin-Furlow Julie
Department of Microbiology and Cell Science, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, FL, USA.
Genetics Institute, University of Florida, Gainesville, FL, USA.
bioRxiv. 2025 Jul 3:2025.07.03.662939. doi: 10.1101/2025.07.03.662939.
is a halophilic archaeon that preferentially utilizes glycerol as a carbon source, placing glycerol kinase (GK, ) at the center of its metabolism. In contrast to bacterial GKs, which are often regulated by allosteric inhibition, GK lacks this mode of control, indicating alternative regulatory mechanisms. Here, we show that lysine acetylation of 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 Δ 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.
是一种嗜盐古菌，优先利用甘油作为碳源，这使得甘油激酶（GK）处于其新陈代谢的中心位置。与通常受变构抑制调节的细菌GK不同，该GK缺乏这种控制模式，表明存在其他调节机制。在这里，我们表明，GK的赖氨酸乙酰化在甘油上生长期间增强了其活性和丰度，其中K153被确定为主要修饰位点。结构建模和比较基因组学表明，K153位于嗜盐古菌GK共有的保守柔性环中。通过AQUA-MS测定，碳源从葡萄糖转变为甘油导致K153乙酰化形式的活性增加和富集。GK和乙酰化模拟物K153Q支持在甘油上生长，而非乙酰化的K153R变体则不能。热迁移分析表明，K153R取代降低了GK的稳定性，而K153Q则没有影响。尺寸排阻色谱表明，GK主要是二聚体，但从甘油培养的细胞中纯化并用甘油测定时会形成四聚体，这与最高的K153乙酰化水平一致。动力学分析表明，K153乙酰化是维持协同底物结合所必需的，非乙酰化的K153R变体表现出变构行为丧失。发现GNAT家族乙酰转移酶Pat2在K153处使GK乙酰化，Δ突变体表现出GK蛋白丰度降低，将Pat2与GK的调节联系起来。这些结果确定了一种动态的、碳源响应性的赖氨酸乙酰化机制，该机制调节GK，突出了赖氨酸乙酰化作为嗜盐古菌代谢调节的关键组成部分。
