Abstract
Lysine acetylation has emerged as a global protein regulation system in all domains of life. Sirtuins, or Sir2-like enzymes, are a family of histone deacetylases characterized by their employing NAD+ as a co-substrate. Sirtuins can deacetylate several acetylated proteins, but a consensus substrate recognition sequence has not yet been established. Product inhibition of many eukaryotic sirtuins by nicotinamide and its analogues has been studied in vitro due to their potential role as anticancer agents. In this work, the kinetics of CobB, the main Escherichia coli deacetylase, have been characterized. To our knowledge, this is the first kinetic characterization of a sirtuin employing a fully acetylated and natively folded protein as a substrate. CobB deacetylated several acetyl-CoA synthetase acetylated lysines with a single kinetic rate. In addition, in vitro nicotinamide inhibition of CobB has been characterized, and the intracellular nicotinamide concentrations have been determined under different growth conditions. The results suggest that nicotinamide can act as a CobB regulator in vivo. A nicotinamidase deletion strain was thus phenotypically characterized, and it behaved similarly to the ΔcobB strain. The results of this work demonstrate the potential regulatory role of the nicotinamide metabolite in vivo.
Highlights
Protein lysine acetylation is a post-translational modification (PTM) in which the scientific community is increasingly interested [1,2,3]
Acetyl-CoA synthetase protein was purified from an E. coli BL21 (DE3) ΔcobB strain to achieve a high acetylCoA synthetase (Acs) acetylation level
We knew that Acs deacetylation was being partially inhibited, likely by nicotinamide reaction product
Summary
Protein lysine acetylation is a post-translational modification (PTM) in which the scientific community is increasingly interested [1,2,3]. Lysine acetylation consists of the transfer of an acetyl group from a donor molecule such as acetyl-CoA or acetyl-phosphate to a lysine amino acid of a protein This transfer can be catalysed by an acetyltransferase enzyme (KAT) [4,5] or occur in a non-enzymatic or chemical manner [6,7]. The number of proteins that are modified post-translationally by acetylation is continuously discovered in all types of organisms. This PTM has traditionally been associated with transcriptional regulation (histone modifications) [8], in the past 10 years, many metabolic routes have been demonstrated to be regulated by lysine acetylation [9,10,11,12,13,14,15].
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