Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Venkat, Sumana; Chen, Hao; Stahman, Alleigh; Hudson, Denver; McGuire, Paige; Gan, Qinglei; Fan, Chenguang

doi:10.1016/j.jmb.2018.04.031

Cited by 43 publications

(58 citation statements)

References 84 publications

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“…Indeed, the wild-type CS overexpressed in BL21(DE3) cells had no detectable acetylation ( Fig. 1), consistent with our previous studies on MDH and ICD which also showed that wild-type MDH and ICD purified from BL21(DE3) cells had no detectable acetylation [14,15]. To facilitate the purification process and remove truncated proteins due to the usage of the UAG stop codon as the signal for AcK incorporation, we fused a His 6 -tag to the C terminus of the CS.…”

Section: Generating Site-specifically Acetylated Cs Variantssupporting

confidence: 90%

“…Site‐directed mutagenesis of lysine to glutamine has been used commonly to study protein acetylation. However, our previous study showed that such method sometimes could draw opposite conclusions with those derived from the approach of direct incorporation of acetyllysine (AcK) . So in this study, we utilized an optimized system to produce purely acetylated CS variants at specific sites listed above by the genetic code expansion strategy individually .…”

Section: Resultsmentioning

confidence: 99%

“…Acetylome studies have identified multiple lysine acetylation sites in the E. coli Type II CS , but their functions are still unknown. Our group has studied the lysine acetylation of two TCA cycle enzymes, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH), and showed that lysine acetylation had different effects on these two TCA enzymes . In this study, we characterized lysine acetylation of the E. coli Type II CS and explored its modification processes.…”

Section: Introductionmentioning

confidence: 99%

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Characterizing lysine acetylation of Escherichia coli type II citrate synthase

et al. 2019

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The citrate synthase (CS) catalyzes the first reaction of the tricarboxylic acid cycle, playing an important role in central metabolism. The acetylation of lysine residues in the Escherichia coli Type II CS has been identified at multiple sites by proteomic studies, but their effects remain unknown. In this study, we applied the genetic code expansion strategy to generate 10 site‐specifically acetylated CS variants which have been identified in nature. Enzyme assays and kinetic analyses showed that lysine acetylation could decrease the overall CS enzyme activity, largely due to the acetylation of K295 which impaired the binding of acetyl‐coenzyme A. Further genetic studies as well as in vitro acetylation and deacetylation assays were performed to explore the acetylation and deacetylation processes of the CS, which indicated that the CS could be acetylated by acetyl‐phosphate chemically, and be deacetylated by the CobB deacetylase.

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Section: Generating Site-specifically Acetylated Cs Variantssupporting

confidence: 90%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterizing lysine acetylation of Escherichia coli type II citrate synthase

et al. 2019

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“…Of the two central-metabolic enzymes showing different patterns of acetylation during growth on glucose versus xylose, acetylation does not appear to have any regulatory role, indicating that this posttranslational modification does not always have a regulatory role. To date, only a few enzymes have been shown to be sensitive to lysine acetylation (4,(6)(7)(8), and the vast majority of acetylated lysines likely have no effect on protein function or activity. That being said, it is also clear that some enzymes are sensitive to acetylation and, in these regards, have coopted this promiscuous modification for regulatory roles.…”

Section: Resultsmentioning

confidence: 99%

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

et al. 2019

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Lysine acetylation is thought to provide a mechanism for regulating metabolism in diverse bacteria. Indeed, many studies have shown that the majority of enzymes involved in central metabolism are acetylated and that acetylation can alter enzyme activity. However, the details regarding this regulatory mechanism are still unclear, specifically with regard to the signals that induce lysine acetylation. To better understand this global regulatory mechanism, we profiled changes in lysine acetylation during growth of Escherichia coli on the hexose glucose or the pentose xylose at both high and low sugar concentrations using label-free mass spectrometry. The goal was to see whether lysine acetylation differed during growth on these two different sugars. No significant differences, however, were observed. Rather, the initial sugar concentration was the principal factor governing changes in lysine acetylation, with higher sugar concentrations causing more acetylation. These results suggest that acetylation does not target specific metabolic pathways but rather simply targets accessible lysines, which may or may not alter enzyme activity. They further suggest that lysine acetylation principally results from conditions that favor accumulation of acetyl phosphate, the principal acetate donor in E. coli. IMPORTANCE Bacteria alter their metabolism in response to nutrient availability, growth conditions, and environmental stresses using a number of different mechanisms. One is lysine acetylation, a posttranslational modification known to target many metabolic enzymes. However, little is known about this regulatory mode. We investigated the factors inducing changes in lysine acetylation by comparing growth on glucose and xylose. We found that the specific sugar used for growth did not alter the pattern of acetylation; rather, the amount of sugar did, with more sugar causing more acetylation. These results imply that lysine acetylation is a global regulatory mechanism that is responsive not to the specific carbon source per se but rather to the accumulation of downstream metabolites.A n abundant posttranslational modification in many bacteria is N -lysine acetylation (1,2). Multiple studies have shown that lysine acetylation predominantly targets the enzymes involved in central metabolism (2)(3)(4)(5). Because some of these lysines are catalytically active, their acetylation may regulate metabolism in bacteria. This hypothesis is supported by several in vitro studies showing that lysine acetylation indeed alters the activity of some enzymes involved in central metabolism (4, 6-8). However, it is still not clear what role lysine acetylation plays in regulating metabolism. One compelling model is that lysine acetylation provides a global mechanism by which cells regulate metabolism in response to their energy status. The response to energy status occurs Citation Schilling B,

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“…Besides eukaryotes, more and more proteomic studies have shown that lysine acetylation is also widely distributed in prokaryotic cells, enriched in metabolic pathways and protein biosynthesis [ 54 ]. Recently, our group has applied the genetic code expansion in studying two metabolic enzymes, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) of E. coli [ 55 , 56 ]. Interestingly, although both enzymes are in the tricarboxylic acid (TCA) cycle, the effects of lysine acetylation are completely different: acetylation of MDH increased the enzyme activity, while acetylation of ICDH decreased the enzyme activity.…”

Section: Lysine Acetylation and Its Analogs By Genetic Code Expansmentioning

confidence: 99%

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

et al. 2018

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Nowadays advanced mass spectrometry techniques make the identification of protein posttranslational modifications (PTMs) much easier than ever before. A series of proteomic studies have demonstrated that large numbers of proteins in cells are modified by phosphorylation, acetylation and many other types of PTMs. However, only limited studies have been performed to validate or characterize those identified modification targets, mostly because PTMs are very dynamic, undergoing large changes in different growth stages or conditions. To overcome this issue, the genetic code expansion strategy has been introduced into PTM studies to genetically incorporate modified amino acids directly into desired positions of target proteins. Without using modifying enzymes, the genetic code expansion strategy could generate homogeneously modified proteins, thus providing powerful tools for PTM studies. In this review, we summarized recent development of genetic code expansion in PTM studies for research groups in this field.

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Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

Cited by 43 publications

References 84 publications

Characterizing lysine acetylation of Escherichia coli type II citrate synthase

Characterizing lysine acetylation of Escherichia coli type II citrate synthase

Global Lysine Acetylation in Escherichia coli Results from Growth Conditions That Favor Acetate Fermentation

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies

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