Abstract
Understanding phosphorylation-mediated regulation of metabolic enzymes, pathways, and cell phenotypes under metabolic shifts represents a major challenge. The kinases associated with most phosphorylation sites and the link between phosphorylation and enzyme activity remain unknown. In this study, we performed stable isotope labeling by amino acids in cell culture (SILAC)-based proteome and phosphoproteome analysis of Escherichia coli ΔyeaG, a strain lacking a poorly characterized serine/threonine kinase YeaG, to decipher kinase-substrate interactions and the effects on metabolic phenotype during shifts from glucose to malate. The starting point of our analysis was the identification of physiological conditions under which ΔyeaG exhibits a clear phenotype. By metabolic profiling, we discovered that ΔyeaG strain has a significantly shorter lag phase than the wild type during metabolic shift from glucose to malate. Under those conditions, our SILAC analysis revealed several proteins that were differentially phosphorylated in the ΔyeaG strain. By focusing on metabolic enzymes potentially involved in central carbon metabolism, we narrowed down our search for putative YeaG substrates and identified isocitrate lyase AceA as the direct substrate of YeaG. YeaG was capable of phosphorylating AceA in vitro only in the presence of malate, suggesting that this phosphorylation event is indeed relevant for glucose to malate shift. There is currently not enough evidence to firmly establish the exact mechanism of this newly observed regulatory phenomenon. However, our study clearly exemplifies the usefulness of SILAC-based approaches in identifying proteins kinase substrates, when applied in physiological conditions relevant for the activity of the protein kinase in question.
Highlights
Metabolic adaptation is one of the major bacterial responses for coping with the changing environment (Liebeke and Lalk, 2014; Goo et al, 2015)
We compared the growth of the ∆yeaG strain to that of the wild type E. coli during metabolic shifts from glucose to various other carbon sources
The most striking phenotype of ∆yeaG was a significant reduction of the duration of lag phase upon transition from growth on glucose to growth on malate (Figure 1B)
Summary
Metabolic adaptation is one of the major bacterial responses for coping with the changing environment (Liebeke and Lalk, 2014; Goo et al, 2015). Faster mechanism of response is the modification of activity of enzymes already present in the bacterial cell This is typically achieved by allosteric regulation (Xu et al, 2012; Link et al, 2013) or reversible post-translational modifications (PTMs; Nussinov et al, 2012; Macek et al, 2019) of bacterial proteins. Bacterial metabolic adaptation processes are known to depend on histidine-kinases and response regulators of the so-called two component systems (Charbonnier et al, 2017; Quiroz-Rocha et al, 2017; GómezMejia et al, 2018). A major metabolic switch in bacteria, is known to critically depend on histidine and cysteine phosphorylation of components of the phosphoenolpyruvate:carbohydrate phosphotransferase system (Deutscher et al, 2014). Bacterial protein-tyrosine and -serine/threonine kinases are known to regulate the metabolism by directly phosphorylating transcription regulators and modifying their affinity for binding of target DNA sequences (Derouiche et al, 2013, 2015; Kobir et al, 2014)
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