Protein lysine methylation is a prevalent post-translational modification (PTM) and plays critical roles in all domains of life. However, its extent and function in photosynthetic organisms are still largely unknown. Cyanobacteria are a large group of prokaryotes that carry out oxygenic photosynthesis and are applied extensively in studies of photosynthetic mechanisms and environmental adaptation. Here we integrated propionylation of monomethylated proteins, enrichment of the modified peptides, and mass spectrometry (MS) analysis to identify monomethylated proteins in Synechocystis sp. PCC 6803 (Synechocystis). Overall, we identified 376 monomethylation sites in 270 proteins, with numerous monomethylated proteins participating in photosynthesis and carbon metabolism. We subsequently demonstrated that CpcM, a previously identified asparagine methyltransferase in Synechocystis, could catalyze lysine monomethylation of the potential aspartate aminotransferase Sll0480 both in vivo and in vitro and regulate the enzyme activity of Sll0480. The loss of CpcM led to decreases in the maximum quantum yield in primary photosystem II (PSII) and the efficiency of energy transfer during the photosynthetic reaction in Synechocystis. We report the first lysine monomethylome in a photosynthetic organism and present a critical database for functional analyses of monomethylation in cyanobacteria. The large number of monomethylated proteins and the identification of CpcM as the lysine methyltransferase in cyanobacteria suggest that reversible methylation may influence the metabolic process and photosynthesis in both cyanobacteria and plants.
研究问题：蓝细菌中发生赖氨酸甲基化的蛋白有哪些？是否有赖氨酸甲基转移酶？赖氨酸甲基化有何功能？研究方法：在本研究中，我们通过对单甲基化蛋白质进行化学丙酰化，将赖氨酸单甲基化与赖氨酸二甲基化和三甲基化区分开。利用免疫亲和的方法进行单甲基化修饰肽段的富集，最后利用高精度质谱鉴定技术，实现了集胞藻PCC6803赖氨酸单甲基化蛋白的系统鉴定，共包括376个赖氨酸单甲基化位点，分布于270个蛋白质中。GO功能富集和KEGG通路分析提示赖氨酸甲基化对光合作用及能量代谢的调控作用。对蓝细菌全基因序列进行保守结构域比对，预测可能的赖氨酸甲基转移酶。结合基因敲除以及体外甲基化反应验证CpcM是一个新的赖氨酸甲基转移酶。对CpcM敲除株和野生型进行质谱检测找到CpcM的作用底物Sll0480。体外甲基化反应联合质谱检测技术对Sll0480被CpcM甲基化的靶位点进行精准定位。本研究揭示CpcM催化的蛋白质赖氨酸甲基化对转氨酶活性的调控机制，以及对光合作用的潜在影响。主要结果1：获得光合生物中首个赖氨酸单甲基化蛋白组数据。主要结果2：在模式蓝细菌集胞藻PCC6803中发现首个赖氨酸甲基转移酶CpcM。主要结果3：CpcM通过催化Sll0480赖氨酸甲基化调控底物蛋白的天冬氨酸转氨酶活性。主要结果4：赖氨酸甲基转移酶CpcM的敲除影响了蓝细菌光合作用电子传递。数据链接：原始质谱文件及谱图：http://www.peptideatlas.org/PASS/PASS01127





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