Characterizing lysine acetylation of Escherichia coli type II citrate synthase.

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.

Genome-Wide Quantification of the Effect of Gene Overexpression on Escherichia coli Growth.

Recombinant protein production plays an essential role in both biological studies and pharmaceutical production. Escherichia coli is one of the most favorable hosts for this purpose. Although a number of strategies for optimizing protein production have been developed, the effect of gene overexpression on host cell growth has been much less studied. Here, we performed high-throughput tests on the E. coli a complete set of E. coli K-12 ORF archive (ASKA) collection to quantify the effects of overexpressing individual E. coli genes on its growth. The results indicated that overexpressing membrane-associated proteins or proteins with high abundances of branched-chain amino acids tended to impair cell growth, the latter of which could be remedied by amino acid supplementation. Through this study, we expect to provide an index for a fast pre-study estimate of host cell growth in order to choose proper rescuing approaches when working with different proteins.

Recent Development of Genetic Code Expansion for Posttranslational Modification Studies.

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.

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli.

The Escherichia coli isocitrate dehydrogenase (ICDH) is one of the tricarboxylic acid cycle enzymes, playing key roles in energy production and carbon flux regulation. E. coli ICDH was the first bacterial enzyme shown to be regulated by reversible phosphorylation. However, the effect of lysine acetylation on E. coli ICDH, which has no sequence similarity with its counterparts in eukaryotes, is still unclear. Based on previous studies of E. coli acetylome and ICDH crystal structures, eight lysine residues were selected for mutational and kinetic analyses. They were replaced with acetyllysine by the genetic code expansion strategy or substituted with glutamine as a classic approach. Although acetylation decreased the overall ICDH activity, its effects were different site by site. Deacetylation tests demonstrated that the CobB deacetylase could deacetylate ICDH both in vivo and in vitro, but CobB was only specific for lysine residues at the protein surface. On the other hand, ICDH could be acetylated by acetyl-phosphate chemically in vitro. And in vivo acetylation tests indicated that the acetylation level of ICDH was correlated with the amounts of intracellular acetyl-phosphate. This study nicely complements previous proteomic studies to provide direct biochemical evidence for ICDH acetylation.