Modulation of the bacterial CobB sirtuin deacylase activity by N-terminal acetylation Anastacia R. Parksa and Jorge C. Escalante-Semerenaa,1 aDepartment of Microbiology, University of Georgia, Athens, GA 30606 Edited by John E. Cronan, University of Illinois at Urbana–Champaign, Urbana, IL, and approved May 20, 2020 (received for review March 23, 2020) In eukaryotic cells, the N-terminal amino moiety of many proteins acetylated in bacteria, such as secretion chaperone SecB of is modified by N-acetyltransferases (NATs). This protein modifica- Escherichia coli and the virulence factor ESAT-6 (ExsA) of several tion can alter the folding of the target protein; can affect binding Mycobacterium species. In the case of the ESAT-6 (ExsA) protein, interactions of the target protein with substrates, allosteric effec- acetylation of its N terminus abolished binding interactions with its tors, or other proteins; or can trigger protein degradation. In pro- protein partner CFP10 (ExsB) and attenuated Mycobacterium karyotes, only ribosomal proteins are known to be N-terminally marinum virulence (17–19). Even though there has been identi- acetylated, and the acetyltransferases responsible for this modifi- fication of N-terminal acetylation in prokaryotes, the acetyl- cation belong to the Rim family of proteins. Here, we report that, transferases responsible for the modifications have not been in Salmonella enterica, the sirtuin deacylase CobB long isoform identified. For example, recently published bacterial N-terminal (CobBL) is N-terminally acetylated by the YiaC protein of this bac- acetylomes of Pseudomonas aruginosa, Acinetobacter baumannii, terium. Results of in vitro acetylation assays showed that CobBL and M. tuberculosis showed that roughly ∼10% of the proteins was acetylated by YiaC; liquid chromatography-tandem mass spec- were N-terminally acetylated (8, 11, 20), but the enzymes re- trometry (LC-MS/MS) was used to confirm these results. Results of sponsible for such modifications were not identified. in vitro and in vivo experiments showed that CobBL deacetylase The Salmonella enterica genome contains ∼26 GNATs, but the activity was negatively affected when YiaC acetylated its N termi- function of about a third of them has yet to be defined. In ad- nus. We report 1) modulation of a bacterial sirtuin deacylase ac- dition, the S. enterica genome possesses one nicotinamide ade- tivity by acetylation, 2) that the Gcn5-related YiaC protein is the nine dinucleotide (NAD+)-dependent sirtuin deacylase CobB, acetyltransferase that modifies CobBL, and 3) that YiaC is an NAT. Based whose function works in concert with the protein acetyl- MICROBIOLOGY on our data, we propose the name of NatA (N-acyltransferase A) in lieu transferase (Pat) enzyme to reversibly modulate the activity of of YiaC to reflect the function of the enzyme. acetyl-CoA synthetase (Acs) (21, 22). As shown in (Scheme 1, the CobB-dependent deacetylation reaction consumes NAD+ sirtuin | CobB sirtuin deacylase | posttranslational modification | N-terminal and yields O-acetyl-ADP (adenosine diphosphate ribose) and acetylation | bacterial GNAT nicotinamide. Interestingly, this bacterium synthesizes two biologically active rotein acylation is common in prokaryotes and eukaryotes, isoforms of CobB, referred to as CobBS (CobB short isoform) Pand it is an effective and rapid means of controlling protein and CobBL (CobB long isoform), which differ in size by a function in response to diverse stimuli (1, 2). What stands out 37-residue N-terminal extension of the catalytic core (Fig. 1). about protein acylation is the diversity of organic acids used by Our group showed that both isoforms of CobB deacetylated cells to modify proteins (e.g., acetate, propionate, malonate, their bona fide protein substrate (i.e., acetylated acetyl-CoA succinate, etc.) and the large number of acyltransferases that synthetase [AcsAc]) in vivo and in vitro (23). However, the catalyze the modifications (1). Many of the acyltransferases that physiological relevance of the two CobB isoforms in S. enterica is modify proteins and small molecules belong to the protein su- unknown. Here, we report that the CobBL sirtuin deacylase perfamily PF00583, and among this family, many proteins con- isoform of this bacterium is N-terminally acetylated and that the tain the so-called GNAT (GCN5-related N-acetyltransferase) putative YiaC acetyltransferase acetylates the N terminus of domain (IPR000182). GNATs acylate free amino groups of proteins or small molecules (1, 2). For example, there is a subset Significance of well-studied GNATs that modify the e amino group (Ne)in α lysine side chains (3, 4), while other GNATs modify the amino N-terminal protein acetylation is poorly understood in bacteria. α – group (N ) of the starting residue of proteins (5 8). Herein, we report the identification of an Nα acetyltransferase To frame the work reported here, we note that many eukaryotic (NAT) that modulates the activity of a sirtuin deacylase in a hu- proteins are acetylated on their N termini and that the acetyl- man pathogen. This is significant because the alluded enzyme transferases responsible for these modifications are referred to as (named N-acyltransferase A [NatA], formerly YiaC) is a prokaryotic N-acetyltransferases (NATs). In general, NATs catalyze the non-Rim–type NAT, and N-terminal acetylation of a bacterial sir- transfer of the acetyl group from acetyl-Coenzyme A (AcCoA) to tuin has not been reported. Also significant is the fact that NatA a primary amine of a small molecule or the N-terminal amino affects the metabolism of acetate, a short-chain fatty acid known α group of a peptide or protein. In eukaryotes, N acetylation has to play an important role in pathogenesis in the human gut. been suggested to alter protein folding, protein–protein interac- tions, and protein degradation (9–12). In contrast, little is known Author contributions: A.R.P. and J.C.E.-S. designed research; A.R.P. performed research; about the enzymes that catalyze N-terminal protein acetylation in A.R.P. and J.C.E.-S. analyzed data; J.C.E.-S. conceptualized the project; and A.R.P. and prokaryotes and what the physiological reasons for such modifi- J.C.E.-S. wrote the paper. cation may be. Examples of N-terminal acetylation of bacterial The authors declare no competing interest. proteins, where the acetyltransferase is known, are the acetylation This article is a PNAS Direct Submission. of ribosomal proteins S18, S5, and L7/L12 by acetyltransferases Published under the PNAS license. RimI, RimJ, and RimL (13–15). These acetyltransferases were 1To whom correspondence may be addressed. Email: [email protected]. thought to have high substrate specificity until recently, when the This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Mycobacterium tuberculosis RimI was shown to acetylate different doi:10.1073/pnas.2005296117/-/DCSupplemental. peptides in vitro (16). Several other proteins are known to be Nα www.pnas.org/cgi/doi/10.1073/pnas.2005296117 PNAS Latest Articles | 1of7 Downloaded by guest on September 27, 2021 Scheme 1. CobBL. We also report in vivo and in vitro evidence that YiaC- dependent N-terminal acetylation of CobBL negatively affects its deacetylase activity. Results YiaC Acetylates CobBL but Not CobBS. A search for protein sub- strates for the S. enterica putative GNATs led us to discover that the S. enterica YiaC protein acetylated S. enterica CobBL but not CobBS. As shown in Fig. 2, when both isoforms of S. enterica CobB were incubated with [1-14C]-AcCoA as a function of YiaC, radiolabel was transferred to CobBL but not to CobBS (Fig. 2, lanes 4 and 5, respectively). Since CobBS was not acetylated, we surmised that the sites of acetylation were located within the 37-amino acid N-terminal, arginine-rich motif of CobBL (Fig. 1). YiaC Does Not Acetylate Ne Amino Groups of Lysine Residues. The N-terminal extension of CobBL contains two lysines (K14, K16), which we investigated as possible acetylation sites. To test this possibility, we changed K14 and K16 to alanine, independently K14A K16A and in combination. The three variants (CobBL , CobBL , K14A,K16A and CobBL ) were overproduced, isolated, and incu- bated with YiaC in the presence of [1-14C]-AcCoA. Surprisingly, YiaC acetylated all three CobBL variants (Fig. 3, lanes 5, 7, and 9), suggesting that, under the assay conditions used, YiaC did not modify the Ne position of either K14 or K16. We note that the K14A,K16A intensity of the signal for acetylated CobBL variant was less than the single-amino acid variants and that the signal in- tensity was commensurate to the amount of protein loaded on K14A,K16A the gel. The yield of CobBL variant was lower than those of the single-amino acid variants. (Fig. 5, lanes 2 and 8) but did not acetylate the CobBL peptide Ac whose first residue was L-Met (Fig. 5, lane 5). Collectively, these YiaC Modifies the N Terminus of CobBL In Vitro. To support our data showed that YiaC was an Nα protein acetyltransferase (NAT). hypothesis that CobBL is Nα acetylated, liquid chromatography- tandem mass spectrometry (LC-MS/MS) was conducted. Results of peptide fingerprinting analysis of acetylated CobB long iso- Ac form ( CobBL) unequivocally showed that the N terminus of CobBL was acetylated by YiaC (Fig. 4). To confirm the LC-MS/MS data, two peptides of the first 50 amino acids of CobBL were synthesized (Peptide 2.0, Virginia): one started with unmodified L-Met, and the second one started with Ac L-Met . In vitro