Optimizing Kinetic Assay Conditions to Study Bacterial Protein Lysine Acetyltransferases in Escherichia coli

Abstract

Protein acetylation is a widespread post translational modification that can be observed in prokaryotes. Recent studies have shown that a multitude of bacterial proteins undergo two types of lysine acetylation: non‐enzymatic and enzymatic. Non‐enzymatic acetylation via the small molecule acetyl‐phosphate (AcP) has been the predominant focus of prior studies. However, recent work indicates that enzymatic protein acetylation by a set of Ne‐lysine acetyltransferases (KATs) in Escherichia coli is extensive and offers new opportunities to more deeply study the role of enzymatic protein acetylation in bacteria. One of the barriers to studying KAT acetylation is that current enzymatic acetylation methods do not appear to consider self‐acetylation properties of some KATs, which can lead to inconclusive results. Thus, finding optimal conditions for controlling or accounting for KAT self‐acetylation is vital to understand its role in enzymatic acetylation of substrate proteins. Therefore, we have examined the self‐acetylation properties of YfiQ and its effect on acetylating a previously uncharacterized protein substrate. We found YfiQ is already acetylated after heterologous expression and purification, which complicates interpretation of enzymatic assay results. Therefore, we are currently optimizing our system to account for this characteristic and are exploring different reaction conditions to study the enzymatic acetylation of the previously uncharacterized protein more deeply.Support or Funding InformationResearch reported in this work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM133506 (to MLK).