a-facile-protocol-to-generate-site-specifically-acetylated-proteins

A Facile Protocol to Generate Site-Specifically Acetylated Proteins in <em>Escherichia Coli</em>

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. ...

University of Arkansas

Protein acetylation plays important roles in many biological processes. Malate dehydrogenase (MDH), a key enzyme in the tricarboxylic acid cycle, has been identified to be acetylated in bacteria by proteomic studies, but no further characterization has been reported. One challenge for studying protein acetylation is to get purely acetylated proteins at specific positions. Here, we applied the genetic code expansion strategy to site-specifically incorporate Nε-acetyllysine into MDH. The acetylation of lysine residues in MDH could enhance its enzyme activity. The Escherichia coli deacetylase CobB could deacetylate acetylated MDH, while the E. coli acetyltransferase YfiQ cannot acetylate MDH efficiently. Our results also demonstrated that acetyl-CoA or acetyl-phosphate could acetylate MDH chemically in vitro. Furthermore, the acetylation level of MDH was shown to be affected by carbon sources in the growth medium.

Studying the Lysine Acetylation of Malate Dehydrogenase.

Aminoacyl-tRNA synthetases (aaRSs) play essential roles in protein synthesis. As a member of the aaRS family, the tyrosyl-tRNA synthetase (TyrRS) in Escherichia coli has been shown in proteomic studies to be acetylated at multiple lysine residues. However, these putative acetylation targets have not yet been biochemically characterized. In this study, we applied a genetic-code-expansion strategy to site-specifically incorporate Nϵ -acetyl-l-lysine into selected positions of TyrRS for in vitro characterization. Enzyme assays demonstrated that acetylation at K85, K235, and K238 could impair the enzyme activity. In vitro deacetylation experiments showed that most acetylated lysine residues in TyrRS were sensitive to the E. coli deacetylase CobB but not YcgC. In vitro acetylation assays indicated that 25 members of the Gcn5-related N-acetyltransferase family in E. coli, including YfiQ, could not acetylate TyrRS efficiently, whereas TyrRS could be acetylated chemically by acetyl-CoA or acetyl-phosphate (AcP) only. Our in vitro characterization experiments indicated that lysine acetylation could be a possible mechanism for modulating aaRS enzyme activities, thus affecting translation.

Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli.

Reversible lysine acetylation is one of the most widely distributed post-translational modifications; it is involved in a variety of biological processes and can be found in all three domains of life. Acetyltransferases and deacetylases work coordinately to control levels of protein acetylation. In this work, we applied the genetic code expansion strategy to site-specifically incorporate Nε-thioacetyl-l-lysine (TAcK) as an analog of Nε-acetyl-l-lysine (AcK) into green fluorescent protein and malate dehydrogenase in Escherichia coli. We showed that TAcK could serve as an ideal functional mimic for AcK. It could also resist the bacterial sirtuin-type deacetylase CobB. Thus, genetic incorporation of TAcK as a non-deacetylatable analog of AcK into proteins will facilitate in vivo studies of protein acetylation.

Article	Year
Studying the Lysine Acetylation of Malate Dehydrogenase. Journal of molecular biology\| PubMed ID: 28366830	2017
Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli. Chembiochem : a European journal of chemical biology\| PubMed ID: 28741290	2017
Genetically encoding thioacetyl-lysine as a non-deacetylatable analog of lysine acetylation in Escherichia coli. FEBS open bio\| PubMed ID: 29123988	2017

Caroline Gregory

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