Dual lysine and N‐terminal acetyltransferases reveal the complexity underpinning protein acetylation
2020; Springer Nature; Volume: 16; Issue: 7 Linguagem: Inglês
10.15252/msb.20209464
ISSN1744-4292
AutoresWilly V. Bienvenut, Annika Brünje, J. Boyer, Jens S Mühlenbeck, Gautier Bernal, Ines Lassowskat, Cyril Dian, Eric Linster, Trinh V. Dinh, Minna M. Koskela, Vincent Jung, Julian Seidel, Laura K Schyrba, Aiste Ivanauskaite, Jürgen Eirich, Rüdiger Hell, Dirk Schwarzer, Paula Mulo, Markus Wirtz, Thierry Meinnel, Carmela Giglione, Iris Finkemeier,
Tópico(s)Signaling Pathways in Disease
ResumoArticle7 July 2020Open Access Transparent process Dual lysine and N-terminal acetyltransferases reveal the complexity underpinning protein acetylation Willy V Bienvenut orcid.org/0000-0003-4192-3920 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Annika Brünje orcid.org/0000-0002-8979-4606 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Jean-Baptiste Boyer orcid.org/0000-0001-5265-3917 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Jens S Mühlenbeck orcid.org/0000-0003-0204-9580 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Gautier Bernal orcid.org/0000-0002-2253-6397 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Ines Lassowskat orcid.org/0000-0002-3832-4006 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Cyril Dian orcid.org/0000-0002-6349-3901 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Eric Linster orcid.org/0000-0001-7963-1400 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Trinh V Dinh orcid.org/0000-0003-2808-3541 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Minna M Koskela orcid.org/0000-0002-6363-1470 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Vincent Jung orcid.org/0000-0003-0530-1737 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Julian Seidel orcid.org/0000-0002-0435-3845 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Laura K Schyrba orcid.org/0000-0002-0291-9745 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Aiste Ivanauskaite orcid.org/0000-0002-1149-5243 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Jürgen Eirich orcid.org/0000-0003-0963-1872 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Rüdiger Hell orcid.org/0000-0002-6238-4818 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Dirk Schwarzer orcid.org/0000-0002-7477-3319 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Paula Mulo orcid.org/0000-0002-8728-3204 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Markus Wirtz orcid.org/0000-0001-7790-4022 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Thierry Meinnel orcid.org/0000-0001-5642-8637 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Carmela Giglione Corresponding Author [email protected] orcid.org/0000-0002-7475-1558 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Iris Finkemeier Corresponding Author [email protected] orcid.org/0000-0002-8972-4026 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Willy V Bienvenut orcid.org/0000-0003-4192-3920 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Annika Brünje orcid.org/0000-0002-8979-4606 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Jean-Baptiste Boyer orcid.org/0000-0001-5265-3917 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Jens S Mühlenbeck orcid.org/0000-0003-0204-9580 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Gautier Bernal orcid.org/0000-0002-2253-6397 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Ines Lassowskat orcid.org/0000-0002-3832-4006 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Cyril Dian orcid.org/0000-0002-6349-3901 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Eric Linster orcid.org/0000-0001-7963-1400 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Trinh V Dinh orcid.org/0000-0003-2808-3541 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Minna M Koskela orcid.org/0000-0002-6363-1470 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Vincent Jung orcid.org/0000-0003-0530-1737 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Julian Seidel orcid.org/0000-0002-0435-3845 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Laura K Schyrba orcid.org/0000-0002-0291-9745 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Aiste Ivanauskaite orcid.org/0000-0002-1149-5243 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Jürgen Eirich orcid.org/0000-0003-0963-1872 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Rüdiger Hell orcid.org/0000-0002-6238-4818 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Dirk Schwarzer orcid.org/0000-0002-7477-3319 Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany Search for more papers by this author Paula Mulo orcid.org/0000-0002-8728-3204 Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland Search for more papers by this author Markus Wirtz orcid.org/0000-0001-7790-4022 Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany Search for more papers by this author Thierry Meinnel orcid.org/0000-0001-5642-8637 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Carmela Giglione Corresponding Author [email protected] orcid.org/0000-0002-7475-1558 Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France Search for more papers by this author Iris Finkemeier Corresponding Author [email protected] orcid.org/0000-0002-8972-4026 Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany Search for more papers by this author Author Information Willy V Bienvenut1,†,‡, Annika Brünje2,‡, Jean-Baptiste Boyer1, Jens S Mühlenbeck2, Gautier Bernal1,†, Ines Lassowskat2, Cyril Dian1, Eric Linster3, Trinh V Dinh3, Minna M Koskela4,†, Vincent Jung1,†, Julian Seidel5, Laura K Schyrba2, Aiste Ivanauskaite4, Jürgen Eirich2, Rüdiger Hell3, Dirk Schwarzer5, Paula Mulo4, Markus Wirtz3, Thierry Meinnel1, Carmela Giglione *,1 and Iris Finkemeier *,2 1Université Paris-Saclay, CEA, CNRS, Institute for Integrative Biology of the Cell (I2BC), Gif-sur-Yvette, France 2Plant Physiology, Institute of Plant Biology and Biotechnology, University of Muenster, Muenster, Germany 3Centre for Organismal Studies Heidelberg, University of Heidelberg, Heidelberg, Germany 4Department of Biochemistry, Molecular Plant Biology, University of Turku, Turku, Finland 5Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany †Present address: Génétique Quantitative et Évolution, Gif-sur-Yvette, France †Present address: Institute of Plant Sciences Paris-Saclay, Gif-sur-Yvette, France †Present address: Institute of Microbiology, Třeboň, Czech Republic †Present address: Institute IMAGINE, Paris, France ‡These authors contributed equally to this work *Corresponding author. Tel: +33 169829844; E-mail: [email protected] *Corresponding author. Tel: +49 251 8323805; E-mail: [email protected] Mol Syst Biol (2020)16:e9464https://doi.org/10.15252/msb.20209464 [Correction added on 28 September 2020, after first online publication: Projekt Deal funding statement has been added.] PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Protein acetylation is a highly frequent protein modification. However, comparatively little is known about its enzymatic machinery. N-α-acetylation (NTA) and ε-lysine acetylation (KA) are known to be catalyzed by distinct families of enzymes (NATs and KATs, respectively), although the possibility that the same GCN5-related N-acetyltransferase (GNAT) can perform both functions has been debated. Here, we discovered a new family of plastid-localized GNATs, which possess a dual specificity. All characterized GNAT family members display a number of unique features. Quantitative mass spectrometry analyses revealed that these enzymes exhibit both distinct KA and relaxed NTA specificities. Furthermore, inactivation of GNAT2 leads to significant NTA or KA decreases of several plastid proteins, while proteins of other compartments were unaffected. The data indicate that these enzymes have specific protein targets and likely display partly redundant selectivity, increasing the robustness of the acetylation process in vivo. In summary, this study revealed a new layer of complexity in the machinery controlling this prevalent modification and suggests that other eukaryotic GNATs may also possess these previously underappreciated broader enzymatic activities. Synopsis A novel protein acetyltransferase family localized or associated to plant plastids is identified and characterised. These GCN5-related N-acetyltransferases (GNATs) have unique amino acid sequence characteristics and unambiguously possess dual N-α- and ε-lysine acetylation activities. An in silico search for putative plastidial N-terminal and lysine acetyltransferases reveals 10 putative GNAT candidates, showing unique features both at the level of the conserved motifs and key residues. Localization to chloroplasts is confirmed for seven of them, while another one is either associated to chloroplasts or localized within the nucleus. All plastid-associated GNATs display distinct lysine acetyltransferase and relaxed N- terminal acetyltransferase substrate specificities. Inactivation of GNAT2, the plastid GNAT involved in photosynthetic state transitions, results in NTA decreases confined to chloroplast proteins, next to the known decreases on photosynthetic KA target proteins. Introduction Each single genome gives rise to myriads of dynamic proteomes. Protein modifications are mainly responsible for expanding the proteome inventory, playing countless functions important to guarantee full protein functionality (for reviews see Friso & van Wijk, 2015; Giglione et al, 2015; Aebersold & Mann, 2016). Among protein modifications, acetylation is one of the most common and intriguing. Two major types of protein acetylations have been identified thus far: N- α- and ε-lysine acetylation. Both modifications involve the transfer of an acetyl moiety from acetyl-coenzyme A (Ac-CoA), either to the α-amino group of the protein N-terminal amino acid or to the ε-amino group of lysines. However, N-terminal acetylation (NTA) and ε-lysine acetylation (KA) display a number of distinctive features. KA is a tightly regulated, reversible post-translational modification, whereas NTA is considered to be irreversible and to take place mainly co-translationally. In a few cases, this latter modification occurs post-translationally such as on actin by NAA80/NatH, on transmembrane proteins by NAA60/NatF, on hormone peptides or in the maturation of exported proteins during plasmodium infection as well as in plastids of plants (Chang et al, 2008; Dinh et al, 2015 and references in Drazic et al, 2016; Aksnes et al, 2019). Both modifications are observed in all kingdoms of life. However, KA and NTA affect separately only 3–20% of all soluble proteins in prokaryotes and it was surmised that the frequency of these modifications increases with the complexity of the organism (Drazic et al, 2016). In multicellular organisms, KA occurs in the nucleus, cytosol, endoplasmic reticulum, mitochondria, and plastids much more frequently than NTA, which is mostly associated with cytoplasmic proteins (Varland et al, 2015; Linster & Wirtz, 2018). Nonetheless, several reports showed that NTA, together with KA (Hartl et al, 2017), is a widespread modification in chloroplasts, which occurs co-translationally on plastid-encoded proteins as well as post-translationally on a significant fraction of imported nuclear-encoded proteins after the cleavage of their transit peptides (Zybailov et al, 2008; Bienvenut et al, 2011, 2012; Bischof et al, 2011; Huesgen et al, 2013). Although the number of experimentally characterized N- and/or K-acetylated (NTAed and KAed) proteins is continuously increasing, many features of the acetyltransferases that catalyze KA and NTA are much less understood, particularly those that originate from prokaryotes and specifically operate in organelles such as mitochondria and chloroplasts. All known N-terminal-α-acetyltransferases (NATs) belong to the superfamily of general control non-repressible 5 (GCN5)-related N-acetyltransferases (GNAT), whereas the identified lysine acetyltransferases (KATs) are grouped in at least three families: GNAT, MYST, and p300/CBP (Friedmann & Marmorstein, 2013; Montgomery et al, 2015; Drazic et al, 2016). GNAT proteins are characterized by a low overall sequence homology (3–23%) but they display conserved secondary and 3D structures (Vetting et al, 2005). Although the GNAT domain has largely diverged, a general profile has been developed and used to identify proteins belonging to the GNAT superfamily, including NATs and KATs (Vetting et al, 2005; Hulo et al, 2008; Rathore et al, 2016; Salah Ud-Din et al, 2016). In eukaryotes, several cytosolic NAT and KAT complexes are known with distinct substrate specificities, which are conserved throughout eukaryotic evolution (Drazic et al, 2016). The NAT specificity is generally defined by the first two amino acids of the substrates, despite the observed negative influence of distant residues (i.e., the K and P inhibitory effects within positions P′2–P′10) (Arnesen et al, 2009b; Hole et al, 2011; Van Damme et al, 2011). This is different to the identified prokaryotic NATs, which are composed of only a catalytic subunit, and which display restricted or extended substrate specificities in eubacteria and archaea, respectively (Giglione et al, 2015). In contrast to eukaryotic proteins and recent results (Christensen et al, 2018; Reverdy et al, 2018; Carabetta et al, 2019), it was believed that prokaryotic KA played only a minor role and not much attention has been given to the corresponding KAT machinery (for review, see (Christensen et al, 2019a,b)). The consensual knowledge favors distinct enzymes for the acetylation of protein N-termini and lysine residues (Liszczak et al, 2011; Magin et al, 2016). The cytosolic acetyltransferases NAA40, NAA50, and NAA60 have been shown to display weak KA and strong NTA activities (Evjenth et al, 2009; Liu et al, 2009; Chu et al, 2011; Yang et al, 2011; Stove et al, 2016; Armbruster et al, 2020; Linster et al, 2020). However, there still is controversy on whether or not NAA10 might catalyze both reactions (Friedmann & Marmorstein, 2013; Magin et al, 2016). Indeed, several reports suggest that the catalytic subunit of the human and yeast NatA complex (Ard1/NAA10) is able to have both NTA and KA activities on specific substrates (Jeong et al, 2002; Arnesen et al, 2009a,b; Evjenth et al, 2009; Shin et al, 2009; Yoon et al, 2014). However, KA failed to be further confirmed for some of these substrates (Arnesen et al, 2005; Murray-Rust et al, 2006) and in vitro KA of other substrates was shown to be enzyme-independent and simply promoted by increasing concentrations of Ac-CoA (Magin et al, 2016; Aksnes et al, 2019). These studies argued against a role for NAA10 in KA and leave open the question of a double KAT/NAT activity for the same acetyltransferase. Interestingly, a new acetyltransferase has been described in the chloroplast of the model plant Arabidopsis thaliana, and, surprisingly, this enzyme displayed auto-KAT activity in addition to unusual promiscuous NAT activity (Dinh et al, 2015). Besides this first report and the recent identification of the plastid lysine acetyltransferase NSI in the chloroplast (Koskela et al, 2018), the plastid NAT and KAT machineries remain uncharacterized thus far. In the current study, we sought to identify putative Arabidopsis NAT and/or KAT candidates using the PROSITE GNAT-associated profiles and the plastid subcellular localization prediction. Such investigation revealed 10 putative Arabidopsis GNAT proteins. Subcellular localization analyses in Arabidopsis protoplasts confirmed a plastid or plastid-associated localization for only eight of the 10 putative GNATs. Furthermore, by using the recently developed global acetylome profiling approach (Dinh et al, 2015), as well as a quantitative mass spectrometry-based lysine acetylome analysis (Lassowskat et al, 2017), we discovered that six of the eight GNATs display significant dual NAT and KAT activities. The remaining two candidates showed only weak KAT as well as NAT activities on a few substrates. All of the GNATs displaying an NTA activity exhibited extended NAT substrate specificities compared to the cytosolic ones. Proof of concept of the dual activity borne by one member was demonstrated in one of the GNAT knockout mutants where deficit of either acetylation levels was observed on plastid proteins. Altogether, this work identifies a new and widespread dual function for the acetyltransferases, which overturns conventional knowledge in this area, and therefore may have far-reaching implications for the study of acetylation in eukaryotic organisms. Results In silico analyses of the Arabidopsis genome revealed 10 GNAT enzymes with putative plastid localization To identify new acetyltransferases responsible for protein acetylation in plastids, we searched the Arabidopsis genome for proteins, which possess both a GCN5-related N-acetyltransferases (GNAT) domain and a predicted organellar N-terminal transit peptide. Our final database search for putative NATs and KATs converged to 10 candidate proteins (Table EV1 and Dataset EV1). Two of these proteins have recently been identified in plastids of Arabidopsis as NAT (NAA70) and as KAT (NSI) enzymes, respectively (Dinh et al, 2015; Koskela et al, 2018). Because the catalytic activity of these proteins (i.e., whether they transfer acetyl groups to protein N-termini, to internal lysine residues of proteins, or to metabolites) cannot be predicted only from their amino acid sequence, we called these enzymes GNAT1–10 (Fig 1, Table EV1). To get some more insights into the relationship between these diverse types of acetyltransferases, we constructed a phylogenetic distance tree including known GNAT proteins from Arabidopsis, yeast, and Escherichia coli (Figs 1A and EV1). Arabidopsis GNAT1–3 cluster together with known histone-acetyltransferase (HAT) proteins from Arabidopsis and yeast (Fig 1A) and defined a first subtype of GNAT-related sequences (subtype 1, Fig EV1). GNAT4, 5, 6, 7, and 10 are located on a distinct branch (subtype 2, Fig EV1) and finally GNAT8 and GNAT9 group into a third subtype (Fig EV1). Figure 1. Putative organellar KAT and NAT genes from Arabidopsis Phylogenetic tree of GNAT candidates from Arabidopsis thaliana (black letters), Saccharomyces cerevisiae (orange letters), and Escherichia coli (green letters) containing the acetyltransferase Pfam domains (PF0058, PF13302, PF13508, PF13673) (Finn et al, 2006). GNAT family sequences were aligned with ClustalW, and a phylogenetic tree was designed by applying the neighbor-joining method. Bootstrap analysis was performed using 2,000 replicates, whereby the resulting bootstrap values (values ≥ 20) are indicated next to the corresponding branches. The tree-specific topology was tested by maximum parsimony analysis. GNAT candidates with a putative organellar localization (TargetP1.1) were highlighted with a green background and named as GNAT1 to GNAT10 in relation to their position in the phylogenetic tree. Squares, triangles, and circles describe the specific acetylation activity, which was reported in literature. The metabolic activity of GNAT2 corresponds to serotonin acetyltransferase (Lee et al, 2014). Schematic overview of organellar GNATs' secondary structure organization (including AtNAA and EcRiml for comparison). Secondary structural elements of the GNAT candidates were determined using Jpred tools in combination with structure homology models (Swiss-model) and are displayed in red (α-helixes), green (β-strands), and orange (supplementary secondary elements). All candidates were predicted with a mitochondrial or a chloroplastic transit peptide (cTP) using TargetP. The mature form of these candidates is released after the excision of this cTP. Positions of main and secondary Acyl-CoA binding domain (Ac-CoA BD) are shown. C, D, A, and B design the four conserved motifs comprising what is referred as the N-acetyltransferase domain (Dyda et al, 2000). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Phylogenetic tree of NAT/KATs based on protein sequence comparison ClustalW alignment was performed using the homologous protein sequence of NAA10, NAA20, NAA30, NAA40 (except for C. reinhardtii), NAA50, NAA60, GNAT1 (At1g26220), GNAT2 (At1g32070, NSI (Koskela et al, 2018)), GNAT3 (At4g19985), GNAT4 (At2g39000, AtNAA70 (Dinh et al, 2015)), GNAT5 (At1g24040), GNAT6 (At2g06025), GNAT7 (At4g28030), GNAT8 (At2g39020), GNAT9 (At2g04845), and GNAT10 (At1g72030) from Arabidopsis thaliana, Medicago truncatula, Vitis vinifera, Populus trichocarpa, Zea mays, Musa acuminata, Triticum aestivum, Solanum lycopersicum, Chlamydomonas reinhardtii, Oryza sativa, Marchantia polymorpha and displayed in a circular mode using the iTOL tool (https://itol.embl.de). Plastid-associated GNATs are colored in green, while the other two GNATs are shown in purple. Download figure Download PowerPoint Proteins of the GNAT superfamily have an overall low primary sequence similarity. However, all GNAT members display a conserved core of six to seven β-sheets and four α-helixes ordered as β0–β1–α1–α2–β2–β3–β4–α3–β5–α4–β6 (Salah Ud-Din et al, 2016). These secondary structural elements arrange in four conserved motifs (A–D; Fig 1B). The A and B domains are involved in Ac-CoA interaction and acceptor substrate binding, respectively. The C and D domains are involved in protein stability (see for review Salah Ud-Din et al, 2016). Although this pattern fits to most of the members of the GNAT superfamily, some deviations were identified such as the missing α2-helix in the HATs, or additional elements, e.g., the additional α-helix between β1 and β2, in the Enterococcus faecalis aminoglycoside 6′-N-acetyltransferase (Wybenga-Groot et al, 1999). The JPred tools (Drozdetskiy et al, 2015) together with homology models obtained from Swiss-model were used to predict the secondary structural elements of the 10 selected GNATs (Fig 1B). All candidates exhibit the typical GNAT topology with several clear primary sequence divergences from the cytosolic catalytic NAT enzymes (Dataset EV1). Particularly, both β1 and β2 strands are poorly conserved in their sequences. Moreover, the length, number, and position of the α-helices between the N-terminal β1 and β2 strands reveal some dissimilarities among the different GNATs (Fig 1B and Dataset EV1). Because variation in this region can reflect differences in substrate specificity (i.e., allowing the formation of different acceptor substrate binding sites), we anticipated a different substrate specificity for our candidates. In addition, a poor sequence conservation was also retrieved among the different GNATs at the level of the secondary elements forming the binding site for the acceptor substrate (loop between β1- and β2-strands, α4-helix and β7-strand), similar to other members of the GNAT superfamily (Salah Ud-Din et al, 2016). Still a number of residues were found remarkably conserved across all selected GNATs (Dataset EV1). The GNAT Ac-CoA binding domain (BD) is generally located at the N-terminal side of the α3-helix. This specific and crucial domain for the acetyltransferase activity shares sequence homology over species with some similarity to the ATP-BD P-loop. For GNATs, the proposed conserved "P-loop like" sequence is [QR]-x-x-G-x-[GA] (Salah Ud-Din et al, 2016), where x could be any amino acid. An enlarged version of this pattern, including L at position 9, was proposed ([QR]-x-x-G-x-[GA]-x-x-L) for eukaryotic NATs (Rathore et al, 2016) and also observed in Staphylococcus aureus GNAT superfamily members (Srivastava et al, 2014). Surprisingly, Cort and co-workers (Cort et al, 2008) reported a major variation for one of the S. aureus GNAT superfamily (SACOL2532) with a G instead of the expected Q/R residue at position 1. A similar variability was observed by Rathore et al (2016), suggesting possible divergences at position 4. Investigation of the consensus "P-loop like" in the putative GNATs clearly showed unique features with a slight degeneration of the conserved sequence for few of them (Table EV2). To verify whether the divergences observed in the Ac-CoA BD were only species-specific, we performed a larger scale orthologue investigation. This approach confirmed the previously mentioned divergences and highlighted some new conserved sites (Table EV2). It appears that the residue at position 5 and 10 retains some specificity associated with hydrophobic residues including L/I/M/V. From this investigation, we could establish an Ac-CoA BD consensus pattern for each of the putative GNATs and a new enlarged version of this pattern corresponding to [RQ]xxG[LIMV][AG]xx[LIMVF][LIMV] (Table EV2). We also observed that seven of the GNAT candidates possess more than one Ac-CoA BD (Table EV2 and Fig 1B). These duplicated "P-loop like" sequences display a degenerated pattern on the residues at positions 5, 9, and 10 (Table EV2) and are extremely rare in cytoplasmic NATs. Out of these multiple Ac-CoA BD, the most conserved ones (labeled as main Ac-CoA BD) were usually located at the N-terminus of the α3-helix as reported for other GNATs (Fig 1B). Several residues previously shown to be involved in substrate binding and specificity in cytosolic NATs (Liszczak et al, 2011, 2013) were also found to be conserved in some of the GNATs (Dataset EV1). For instance, in HsNAA50 the two catalytic residues Y73 in β4 strand and H112 residue in β5 strand, which are representative of the general base positions in GNAT enzymes (Liszczak et al, 2013), are found in GNAT4, 5, 6, and 7 and, to a lesser extent in GNAT9 and 10, in which the equivalent H112 is replaced by E and Y residues, respectively. Similarly, GNAT8 displays a catalytic dyad equivalent to HsNAA30 at these same positions (Y283 in β4 strand and E321 residue in β5 strand). Interestingly, the β4/β5 catalytic dyad is not conserved in GNAT1, 2, and 3. Indeed, Y73 was replaced by I in GNAT1, T in GNAT2, and S in GNAT3, whereas H112 is replaced by F in GNAT2 and by Y in GNAT1 and 3 sugges
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