An Integrated Analytical Approach for Screening Functional Post-Translational Modification Sites in Metabolic Enzymes

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.