Characterizing a novel bacterial sirtuin protein in an intact protein system

Abstract

Sirtuins, proteins that remove acyl-modifications from lysine side chains, have many important medical and biological applications as they are involved in a wide range of cellular processes such as aging, transcriptional regulation through histone tail modification, and stress resistance. They have been suggested to mitigate damage caused by carbon stress and its associated non-enzymatic modification of protein by reactive acyl-CoA species. Protein hyperacylation has been associated with altered metabolic flux or reduced enzymatic activities. Syntrophus aciditrophicus, a bacterium existing in extreme conditions, displays unusually high levels of protein acylation, leading us to wonder whether sirtuins are present in this system to remove these acyl modifications. Sirtuin homologs have been found in these bacteria by genetic homology, but their substrate specificities and activities have not been studied. We developed a novel mass spectrometry based enzymatic assay to test the range of acyl groups S. aciditrophicus sirtuins could recognize and measured their reaction kinetics. Novel sirtuin homologs (Syn42, Syn1020) from S. aciditrophicus were expressed recombinantly. Insulin was chemically modified with acyl-anhydrides corresponding to the desired acyl modification (e.g. acetic anhydride for acetyl-modified insulin, propionic anhydride for propionyl-modified insulin) as a proxy for an acyl-modified substrate. Sirtuin (10 μM) was added to varyied concentrations of acyl-insulin in ammonium acetate buffer (pH ~7.5), with excess NAD+. The reaction was halted at timepoints 1, 3, 5, 10, and 15 minutes by mixing with acetonitrile 1:1 and ubiquitin was added into the mixture as an internal standard for mass spectrometric quantitation. Assay contents were analyzed by mass spectrometry. We have shown that the novel sirtuin Syn42 acts on several acylations including butyryl, glutaryl, succinyl, and propionyllysines. In addition to determining acyl-group substrate specificity, we were also able to measure Michaelis-Menten kinetics, by plotting time courses of sirtuin enzymatic activity. For glutaryl-modified insulin, the Km was determined to be 16.92 μM and the Kcat was determined to be 0.0022 sec−1, which are comparable to previous measurements of mammalian sirtuin SIRT1. In conclusion, we were able to identify and confirm the activity of novel sirtuin from a bacterial source. We were also able to determine its acyl-group substrate specificity as well as its kinetic properties, demonstrating that S. aciditrophicus sirutins have properties on par with those found in mammalian systems. Support or Funding Information This work is funded by the US Department of Energy (UCLA Institute of Genomics and Proteomics, DE-FC03-02ER63421) and the National Institutes of Health (R01GM085402). MALDI-TOF data shows that the bacterial sirtuins will act on a glutaryl modification Comparing of the ratio of the deacylated insulin intensity to that of ubiquitin over time (or the ratio of acylated insulin intensity to ubiquitin) further displays the enzymatic activity on modified insulin. The shift in the ratio of the modified and demodified insulin can be seen at multiple time points using electrospray mass spectrometry. A time course shows the relative intensities of glutaryl-insulin (200 μM) and ubiquitin as seen over a course of 2 hours. Michaelis-Menten kinetics that have been determined for the sirtuin homolog's activity on glutaryllysine. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.