Cyanobacteria are the oldest prokaryotic photoautotrophic microorganisms and have evolved complicated post-translational modification (PTM) machinery to respond to environmental stress. Lysine 2-hydroxyisobutyrylation (Khib) is a newly identified PTM that is reported to play important roles in diverse biological processes, however, its distribution and function in cyanobacteria have not been reported. Here, we performed the first systematic studies of Khib in a model cyanobacterium Synechococcus sp. strain PCC 7002 (Syn7002) using peptide prefractionation, pan-Khib antibody enrichment, and high-accuracy mass spectrometry (MS) analysis. A total of 1875 high-confidence Khib sites on 618 proteins were identified, and a large proportion of Khib sites are present on proteins in the cellular metabolism, protein synthesis, and photosynthesis pathways. Using site-directed mutagenesis and functional studies, we showed that Khib of glutaredoxin (Grx) affects the efficiency of the PS II reaction center and H₂O₂ resistance in Syn7002. Together, this study provides novel insights into the functions of Khib in cyanobacteria and suggests that reversible Khib may influence the stress response and photosynthesis in both cyanobacteria and plants.

中文翻译：

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蓝藻赖氨酸2-羟基异丁酰化的鉴定及功能分析

蓝细菌是最古老的原核光合自养微生物，并进化出复杂的翻译后修饰（PTM）机制来应对环境压力。赖氨酸2-羟基异丁酰化(Khib)是一种新发现的PTM，据报道在多种生物过程中发挥重要作用，但其在蓝藻中的分布和功能尚未见报道。在这里，我们对模型蓝藻聚球藻中的 Khib 进行了首次系统研究。使用肽预分级、pan-Khib 抗体富集和高精度质谱 (MS) 分析对 PCC 7002 (Syn7002) 菌株进行分析。共鉴定出618个蛋白质上的1875个高置信度Khib位点，其中很大一部分Khib位点存在于细胞代谢、蛋白质合成和光合作用途径的蛋白质上。通过定点诱变和功能研究，我们发现谷氧还蛋白 (Grx) 的 Khib 影响Syn7002 中PS II 反应中心的效率和 H ₂ O ₂抗性。总之，这项研究为蓝藻中 Khib 的功能提供了新的见解，并表明可逆的 Khib 可能影响蓝藻和植物的应激反应和光合作用。