Post-translational modifications (PTMs) are pivotal in the orchestration of diverse physiological and pathological processes. Despite this, the identification of functional PTM sites within the vast amount of data remains challenging. Conventionally, those PTM sites are discerned through labor-intensive and time-consuming experiments. Here, we developed an integrated analytical approach for the identification of functional PTM sites on metabolic enzymes via a screening process. Through gene ontology (GO) analysis, we identified 269 enzymes with lysine 2-hydroxyisobutyrylation (Khib) from our proteomics data set of Escherichia coli. The first round of screening was performed based on the enzyme structures/predicted structures using the TM-score engineer, a tool designed to evaluate the impact of PTM on the protein structure. Subsequently, we examined the influence of Khib on the enzyme–substrate interactions through both static and dynamic analyses, molecular docking, and molecular dynamics simulation. Ultimately, we identified NfsB K181hib and ThiF K83hib as potential functional sites. This work has established a novel analytical approach for the identification of functional protein PTM sites, thereby contributing to the understanding of Khib functions.

中文翻译：

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筛选代谢酶功能性翻译后修饰位点的综合分析方法

翻译后修饰 (PTM) 在协调不同的生理和病理过程中至关重要。尽管如此，在大量数据中识别功能性 PTM 位点仍然具有挑战性。传统上，这些 PTM 位点是通过劳动密集型且耗时的实验来识别的。在这里，我们开发了一种综合分析方法，用于通过筛选过程识别代谢酶上的功能性 PTM 位点。通过基因本体 (GO) 分析，我们从大肠杆菌蛋白质组数据集中鉴定了 269 种具有赖氨酸 2-羟基异丁酰化 (Khib) 的酶。第一轮筛选是根据酶结构/预测结构使用 TM-score 工程师进行的，TM-score 工程师是一种旨在评估 PTM 对蛋白质结构影响的工具。随后，我们通过静态和动态分析、分子对接和分子动力学模拟研究了 Khib 对酶-底物相互作用的影响。最终，我们确定 NfsB K181hib 和 ThiF K83hib 为潜在的功能位点。这项工作建立了一种新的分析方法来识别功能蛋白 PTM 位点，从而有助于理解 Khib 功能。