Abstract
Reversible Nε-lysine acetylation has emerging as an important metabolic regulatory mechanism in microorganisms. Herein, we systematically investigated the site-specific and kinetic characterization of enzymatic (lysine acetyltransferase) and nonenzymatic acetylation (AcP-dependent or Acyl-CoA-dependent), as well as their different effect on activity of metabolic enzyme (AMP-forming acetyl-CoA synthetase, Acs). It was found that Bacillus subtilis acetyl-CoA synthetase (BsAcsA) can be acetylated in vitro either catalytically by lysine acetyltransferase BsAcuA and Ac-CoA (at low concentration), or nonenzymatically by Ac-CoA or AcP (at high concentration). Two distinct mechanisms show preference for different lysine acetylation site (enzymatic acetylation for K549 and nonenzymatic acetylation for K524), and reveal different dynamics of relative acetylation changes at these lysine sites. The results demonstrated that lysine residues on the same protein exhibit different acetylation reactivity with acetyl-phosphate and acetyl-CoA, which was determined by surface accessibility, three-dimensional microenvironment, and pKa value of lysine. Acetyl-CoA synthetase is inactivated by AcuA-catalyzed acetylation, but not by nonenzymatic acetylation.
Highlights
Reversible Nε-lysine acetylation, which can change protein conformation, protein charge, protein stability, protein-protein interactions, protein-DNA binding affinity, or protein localization, is an abundant post-translational modification (PTM) in cells of all domains of life
To determine if acetyl-coenzyme A (Ac-CoA) or acetyl phosphate (AcP) can acetylate protein, we investigated Ac-CoA/ AcP-dependent acetylation using the purified recombinant B. subtilis Ac-CoA synthetase (BsAcsA)
Ac-CoA/ AcP-induced acetylation on Bacillus subtilis acetyl-CoA synthetase (BsAcsA) in a dose-dependent manner was observed by Western immunoblot analysis (Fig. 1)
Summary
Acetyl phosphate or acetyl-coenzyme A directly acetylates acetyl-coenzyme A synthetase. The results demonstrated that acetyltransferase BsAcuA significantly accelerated the acetylation of BsAcsA at low concentration of Ac-CoA (can’t chemically acetylate ε-amino group of a lysine residue). It was found that AcuA-catalyzed acetylation of Acs enzymes resulted in 85–90% decrease in acetyl-CoA synthetase activity compared to the nonacetylated enzymes, whereas Ac-CoA/ AcP-dependent acetylation exerted no effect on AcsA enzyme activity (Fig. 4A). The mutants of K549 (AcsAK549Q, AcsAK549A, and AcsAK549R) were essentially inactive, with 10-20% of the wild-type AcsA activity (Fig. 4C) These results indicated that residue K549 can be acetylated by AcuA-catalyzed acetylation, but not by Ac-CoA/AcP-dependent nonenzymatic acetylation. We found that AcP was unable to acetylate conserved lysine residue K628 of S. erythraea AcsA2, whereas acetylated lysine residue K611 (possible role in binding the acetate substrate), resulted in a 30% decrease in acetyl-CoA synthetase activity[17].
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