Structural basis of the catalytic and allosteric mechanism of bacterial acetyltransferase PatZ

24 PatZ, YfiQ, Acetyltransferase, Allosterism, CryoEM 25 26 27 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint

28 29 GCN5-related N-Acetyltransferases (GNATs) play a crucial role in regulating bacterial metabolism by 30 acetylating specific target proteins. Despite their importance in bacterial physiology, the mechanisms 31 underlying GNATs’ enzymatic and regulatory functions remain poorly understood. In this study, we 32 elucidated the structures of Escherichia coli PatZ, a type I GNAT , and investigated its ligand 33 interactions, catalytic processes, and allosterism. PatZ functions as a homotetramer, with each 34 subunit comprising a catalytic domain and a regulatory domain. Our findings reveal that the regulatory 35 domain is essential for acetyltransferase activity, as it not only induces cooperative conformational 36 changes in the catalytic domain but also directly contributes to the formation of substrate binding 37 pockets. Furthermore, a protein structure-based analysis on the evolution of bacterial GNAT types 38 reveals a distinct pattern of the regulatory domain across phyla, underscoring the regulatory domain’s 39 critical role in responding to cellular energy status. 40 41 SIGNIFICANCE STATEMENT 42 43 Post-translational modifications, particularly acetylation mediated by GCN5 -related N -44 Acetyltransferases (GNATs), play a crucial role in bacterial physiology. Protein acetyltransferase Z 45 (PatZ) is a key GNAT with diverse substrates, essential for understanding the bacterial acetylome. 46 This study employs cryogenic electron microscopy, X-ray crystallography, and biochemical analyses 47 to elucidate the mechanistic regulation of Escherichia coli PatZ. Our high-resolution structures reveal 48 PatZ's homo-tetrameric architecture, with each subunit comprising regulatory and GNAT domains. 49 We characterize ligand-PatZ interactions, demonstrating ligand-induced conformational changes that 50 facilitate allosteric regulation of the catalytic domain. Furthermore, our analyses elucidate the 51 regulatory domain's contribution to substrate binding pocket formation, potentially enhancing 52 substrate specificity. Structure -based phylogenetic analysis provides insights into the evolution of 53 diverse regulatory domains in the GNAT superfamily across bacterial taxonomy. This first visualization 54 of PatZ advances our mechanistic understanding of bacterial physiology, offering novel insights into 55 GNAT-mediated bacterial adaptations. 56 57 HIGHLIGHTS 58 59 - E. coli PatZ forms a homotetramer, with each subunit possessing a GNAT catalytic domain 60 and a regulatory domain. 61 - Cooperative binding of acetyl-CoA to the regulatory domains is a prerequisite for inducing the 62 structural compatibility of the catalytic domain with a substrate. 63 - Diverse regulatory domains in GNATs evolved to adapt to varied metabolic conditions across 64 bacterial taxonomy. 65 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint

66 67 Post-translational modifications (PTMs) play a crucial role in modifying protein structure, stability, 68 activity, and their placement within the cell1–3. Acetylation stands out as one of the predominant PTMs, 69 involving the attachment of an acetyl group to the lysine residues or the protein's N -terminal4. This 70 modification is a widespread phenomenon across all forms of life, facilitated either enzymatically by 71 acetyltransferases or non-enzymatically via high-energy compounds such as acetyl phosphate (AcP) 72 or acetyl-CoA (AcCoA)5–8. In eukaryotes, acetylation affects both histones and non-histone proteins9,10, 73 thereby influencing a broad array of cellular and physiological processes including transcription, phase 74 separation, autophagy, mitosis, cellular differentiation, and neuronal activities 11–16 and disruption in 75 acetylation regulation is linked to cancer development and aging17,18. In prokaryotes, acetylation plays 76 a significant role in regulating metabolism, modifying metabolic fluxes, cell growth, and survival19–21. It 77 also plays a part in the regulation of gene expression by altering the activity of transcription factors 78 according to environmental shifts 22. While the physiological significance of Nε-lysine acetylation is 79 widely recognized in eukaryotes, its biological relevance in bacteria is still emerging. 80 81 Enzymatic acetylation involves the addition of an acetyl group to lysine residues by enzymes known as 82 lysine acetyltransferases (KATs) 23. Based on sequence homology and biochemical characteristics, 83 KATs are classified into several families, including the p300/cAMP response element binding protein 84 binding protein (CBP) family, Moz, Ybf2/Sas3, Sas2, Tip60 (MYST) family, and GCN5 -related N -85 acetyltransferase (GNAT) family 11,24,25. The MYST and p300/CBP families are found exclusively in 86 eukaryotic cells, whereas the GNAT family includes orthologous proteins present in bacteria, 87 eukaryotes, and archaea23. Approximately 65% of the GNAT domain superfamily is found in bacteria26. 88 Recent studies reveal that acetylation significantly impacts bacterial physiology, influencing translation 89 in Escherichia coli27, metabolic flux in Salmonella enterica28, and virulence in Francisella novicida29. 90 These findings underscore the broad and vital impact of acetylation on bacterial physiology, ranging 91 from basic cellular processes to complex pathogenic mechanisms. 92 93 The catalytic GNAT domain is highly conserved across species. However, bacterial GNATs exhibit 94 unique regulatory domains which are diverse in different bacterial species8. These GNATs are classified 95 into five types based on their architecture and arrangement of catalytic GNAT domain and regulatory 96 domains (Figure 1a). Type I and II feature a homologous structure of NDP-forming acyl-CoA synthetase 97 in the regulatory domain, while Type III has a cAMP-binding, ACT (aspartate kinase, chorismate mutase, 98 TyrA) or NADP +-binding protein8. Types IV and V do not possess regulatory domain. Truncation or 99 mutations in these regulatory domains significantly diminished GNAT -mediated catalytic activity 8,30, 100 implying that the regulatory and GNAT domains are functionally interdependent. In particular, protein 101 acetyltransferase Z (PatZ), also known as YfiQ, is one of the most well characterized Type I GNATs, 102 with approximately 80% of its amino acids comprising the regulatory domain, and 20 % GNAT domain. 103 PatZ alters its oligomeric states and activity based on the presence of AcCoA, showing different patterns 104 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint in E. coli and S. enterica31,32. Recent studies about PatZ demonstrate that regulatory domain increases 105 the acetyltransferase activity and that ligand binding to regulatory domain elevates AcCoA affinity to 106 GNAT domain31. However, the precise mechanisms remain unclear. 107 108 In this study, we elucidated the homotetrameric structure of functional PatZ using cryo -electron 109 microscopy (cryoEM). Each subunit of PatZ comprises four structural domains, binding to two AcCoA 110 molecules, one ATP, and one phosphate. The unliganded -PatZ adopts an inactive conformation 111 characterized by a closed substrate binding pocket. Our findings reveal that the regulatory domains 112 undergo conformational changes upon ligand binding, altering their interactions with the GNAT domain. 113 Specifically, the binding of AcCoA to the regulatory domain triggers a cooperative structural 114 rearrangement in the GNAT domain, which facilitates the binding of the AcCoA donor and organizes 115 the substrate in close proximity for catalysis. Furthermore, our analysis suggests that PatZ coevolved 116 with its substrate, with the regulatory domain playing a crucial role in substrate interaction. Additionally, 117 we conducted a phylogenetic analysis of bacterial GNAT -mediated acetyltransferase families, which 118 provides insights into the regulatory domain's role in activating distinct acetyl-PTM responses to various 119 metabolic signatures. 120 121 122

123 124 Overall architecture of E. coli PatZ 125 To examine the biochemical and structural characteristics of PatZ, we purified the E. coli PatZ protein 126 and analyzed its acetyltransferase activity towards purified E. coli AcCoA synthetase (Acs) using 127 western blotting with an acetyl-lysine-specific antibody (Figure 1b). When supplied with AcCoA, PatZ 128 efficiently catalyzed the acetylation of Acs, consistent with findings from previous studies33,34. This PatZ-129 mediated enzymatic reaction is also significantly faster than the AcP-mediated non-enzymatic reaction 130 (Figure 1b)35, confirming that the prepared PatZ is active and functional. 131 132 We then delved into the three -dimensional structure of the unliganded form of PatZ to delineate its 133 molecular architecture and assembly. The protein, with a protomer mass of approximately 100 KDa, 134 predominantly forms a homotetramer in solution, as inferred by size exclusion chromatography (Figure 135 1c). CryoEM imaging of the eluted PatZ protein revealed a tetrameric structure characterized by a 136 diamond-shaped, four -fold symmetric architecture. We then achieved a resolution of 2.52 Å for a 137 consensus map showing the composition of four distinct subunits. These units exhibit variable 138 resolution across the structure's periphery and thus structural features were enhanced by multiple 139 rounds of 3D -classification and variability analysis for each domain (Extended Data Figure 1a -e, 140 Supplementary Table 1). 141 142 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Molecular model was built by chasing the side densities and aligns closely with the cryoEM map, as 143 demonstrated by high -quality statistical validation (Q -score=0.67, Extended Data Figure 1f). Four 144 subunits are positioned in a cross-like arrangement, with a 180-degree rotation relative to adjacent units 145 (Figure 1d, Supplementary Video 1), giving the tetramer lengths of approximately 190 Å and 130 Å 146 along the long and short axes, respectively. Notably each subunit is composed of four distinct structural 147 domains. Reflecting on similarities with NDP -forming AcCoA synthetases (ACDs) 36 and proteins 148 containing GNAT motifs37, we designated these regions accordingly the CoA binding domain (CBD), 149 ligase-CoA domain (LCD), ATP grasp domain (AGD), and GNAT domain (GNATD), sequentially 150 arranged from the N- to the C-terminus (Figure 1e, Supplementary Video 1). 151 152 PatZ presents a propensity to form a stable homotetramer structure, showcasing a sophisticated 153 interaction among four protomers. The interactions exhibited significant contact areas with considerable 154 stabilization energies, notably 3103.4 Å2 with a ΔiG of -28.4 kcal/mol for the lateral interface and 619.5 155 Å2 with a ΔiG of -12.0 kcal/mol for the axial interface. The formation of the lateral interface is achieved 156 through an antiparallel hetero -domain interaction between the CBD and LCD via dominant polar 157 interactions (Figure 1f). Conversely, the axial interface with the CBDs stacking in a back-to-back fashion 158 at a 60-degree angle (Figure 1g). This mode of interaction enhances the stability through symmetric 159 predominant hydrophobic contacts. An attempt to disrupt tetramer form by mutating residues at the 160 axial interface led to significant protein aggregation. Collectively, our investigation into the PatZ protein 161 delineates its acetyltransferase function within a uniquely stable tetrameric architecture. 162 163 Structural basis of ligand binding to PatZ 164 Based on the structural homology with ACD1 36 and GNAT 37 proteins, we have identified four 165 hypothetical ligand binding pockets for two AcCoAs, ATP and phosphate (or AcP). To characterize the 166 relationship between ligand and PatZ, we incubated purified PatZ with ATP, AcCoA, and phosphate, 167 and then analyzed it using cryoEM . We obtained a significantly higher resolution map converging at 168 1.99 Å (Q score=0.77, Extended Data Figure 2a -f, Supplementary Table 1). The overall shape and 169 subunit arrangement of liganded PatZ remain identical but notably, we identified two AcCoA in each 170 protomer, which is one in the N -terminal regulatory domain and the other in GNATD (Figure 2a,b, 171 Supplementary Video 2). We also identified phosphate densities located at each four lateral interfaces 172 between the LCD and CBD derived from different protomers (Figure 2a,b). Of note, we could not 173 visualize ATP in AGD of the cryoEM map, which is potentially reasoned by structural dynamics in the 174 domain. Therefore, we performed complementary X-ray crystallographic experiments for the AGD and 175 confirmed bound ATP in AGD resolution with 2.24 Å (Figure 2a,b, Extended Data Figure 2g, 176 Supplementary Table 2). Collectively, we successfully visualized all possible ligand binding structures. 177 178 AcCoA in catalytic GNATD: Within the PatZ subunits, each GNATD adopts a zigzag configuration, 179 oriented outward to maximize its accessibility and functional flexibility. The structural composition of the 180 GNATD includes five α-helices and eight β-strands, forming a donut-shaped tunnel that facilitates the 181 concurrent orientation of AcCoA and the substrate (Supplementary Video 3). This arrangement is 182 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint pivotal for the enzymatic activity, directing AcCoA and substrate into a productive alignment. The 183 GNATD features a positively charged surface that binds to AcCoA, and a negatively charged surface 184 that assists in positioning acceptor lysine residues. AcCoA is strategically placed within a V -shaped 185 cleft, bordered by β -strands, ensuring precise orientation. The pyrophosphate group of AcCoA is 186 anchored through interactions with the phosphate -coordinating loop (P-loop38, G822-G824) including 187 water-mediated hydrogen bonding and a phosphate group on the ribose, which is secured by K860 188 (Figure 2c). Moreover, the acetate group of AcCoA, crucial for triggering enzymatic reactions, is situated 189 near E809. This residue is identified as a potential catalytic site32 and is in close proximity to the putative 190 acceptor lysine binding site, underscoring its significance in the enzymatic process. 191 192 AcCoA in N-terminal CBD: The PatZ enzyme distinguishes itself through the inclusion of a regulatory 193 domain alongside its catalytic domain, a feature not fully elucidated in terms of its mechanistic role. We 194 visualized a well-defined AcCoA density within the N-terminal CBD (1-285) of PatZ. Notably, the CoA 195 portion of AcCoA is positioned to project outward, while the acetyl group is nestled within the inter -196 subunit cleft. The binding pocket is characterized by its positive charge and an elongated, narrow 197 morphology, tailored to interact with AcCoA's three phosphate groups. This interaction is further 198 reinforced by the pocket's design to accommodate the distinct components of AcCoA through a network 199 of hydrogen bonds including water -mediated interactions (Figure 2d). Particularly, the positively 200 charged residues (K23, R26 and R81) within the pocket engage in electrostatic interactions with 201 AcCoA's phosphate groups, underscoring the specificity and efficiency of the binding process. 202 203 Phosphate located in intersubunit interface: We have identified a significant density in the interface 204 between each CBD and LCD derived from different protomers. Considering the homology with ACD136, 205 this density can be attributed to phosphate. The phosphate binding site is located at the tip of the α -206 helices originating from the LCD and CBD, and is further stabilized by additional loops. The phosphate 207 is spatially adjacent to AcCoA, which resides within the CBD (Figure 2d). Since the phosphate ion is 208 deeply embedded inside the structure, stabilizing a lateral dimer. Of note, this phosphate can be 209 converted to AcP during ATP synthesis pathway for ACD enzyme 39. Through structural analysis, we 210 found that although PatZ's regulatory domain is incapable of enzyme catalysis, it still binds phosphate 211 in the same location as ACD1. 212 213 ATP in ATP grasp domain: The ATP grasp domain (AGD, 451 -709) encompasses the ATP binding 214 pocket. Since the dynamic nature of the AGD complicates the visualization of ATP density in cryoEM. 215 Consequently, X-ray crystallography was employed, successfully confirming the presence of ATP within 216 the AGD's binding pocket at a 2.24 Å resolution. This structure shows that the ATP binding site is 217 nestled in a cleft located in the central AGD, engaging in various interactions with surrounding amino 218 acids (Figure 2e). Despite the presence of 1 mM magnesium during the crystallization process, no 219 electron density map was observed. Instead of a divalent ion, the triphosphate moiety of ATP is 220 stabilized by two histidines (H531, H676) in AGD. 221 222 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint AcCoA binding activates GNATD 223 To understand how AcCoA binding activates catalytic GNATD and the chemical basis of its catalytic 224 mechanism, we compared GNATD structures between the apo- and AcCoA-bound states. Binding of 225 AcCoA to GNATD induces significant spatial changes, particularly in the substrate gating motif (V746-226 T763, hereafter, gating motif), which are located near the substrate access channel and undergo a 227 notable secondary structural transition (Figure 3a, Supplementary video 3). In apo-PatZ, the β-sheet of 228 the gating motif is extended, covering the substrate binding sites of GNATD. However, in the presence 229 of AcCoA, this region transforms into a loop and an α-helix, establishing direct interaction with AcCoA 230 (Figure 3b) and opening a substrate binding pocket (Figure 3c). This structural observation indicates 231 the extended β-sheet of gating motif sterically obstructs the interaction between donor AcCoA and the 232 acceptor substrate. AcCoA binding triggers the rearrangement of the gating motif, removing the physical 233 barrier between AcCoA and the substrate. These structural rearrangements can be crucial to organize 234 the AcCoA donor and the substrate in catalytic proximity. Therefore, we conclude that AcCoA binding 235 is required for GNATD by switching the gating motif into an active structural conformation. 236 237 Based on the active conformation of GNATD, we investigated the critical residues that potentially 238 contribute to enzyme activity and substrate binding. By focusing on the distinctive donut hole shape of 239 the GNATD, we observed that the acetyl group of the acetyl-donor AcCoA is positioned at the center of 240 this hole (Figure 3d), oriented towards the substrate binding site. This region contains several charged 241 residues, and we created alanine mutants for four key amino acids (R754, F756, E809, I846) and 242 compared their enzyme activity to that of the wild -type. The results showed that mutations in any of 243 these four residues led to a significant reduction in enzyme activity (Figure 3e). Based on these 244 functional tests and structural analysis, we confirmed that the acetyl group of AcCoA and the acetyl -245 acceptor lysine are strategically positioned around the donut hole of the GNATD, playing a crucial role 246 in the acetyl-transfer reaction. 247 248 To gain a deeper chemical understanding of the enzyme mechanism, we compared our structure with 249 that of the GNATD of Streptomyces lividans PatA (slPatA), which has been structurally characterized 250 in complex with S. enterica Acs (seAcs) (PDB ID: 4U5Y)40 . The GNATD of slPatA is structurally similar 251 to that of PatZ GNATD, with an RMSD of 1.12 Å (Extended Data Figure 3a). By integrating structural 252 information, we identified the arrangement of key catalytic residues in PatZ, including E809 and the 253 carbonyl oxygens of F810 and I846 (Figure 3f). Notably, these residues are highly conserved within the 254 GNAT family. E809, in particular, is recognized as a crucial catalytic residue (Extended Data Figure 3b). 255 Of note, this glutamate residue can activate a water molecule, which coordinated by E123PatA, carbonyl 256 oxygen of V124PatA, amide nitrogen of E160 and K609 Acs, to remove a proton from the lysine amine 257 group, facilitating a nucleophilic attack on the carbonyl carbon of enzyme -bound AcCoA (Figure 3f). 258 Taken together, the structural similarities between slPatA and E. coli PatZ, coupled with the conserved 259 nature of the catalytic residues, underscore the mechanistic insights into the enzymatic function of PatZ 260 (Figure 3g). These findings imply the critical roles of E809 and the amino acid backbones in facilitating 261 the acetyl-transfer reaction, advancing our understanding of the GNAT family’s catalytic mechanisms. 262 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 263 Allosteric regulation for PatZ 264 Next, to investigate the functional and mechanistic association between the regulatory domain and 265 catalytic GNATD, we generated domain-wise truncated PatZ mutants and examined their activity. The 266 AGD-truncated mutant (trAGD) produced soluble aggregates, while the other truncated mutants 267 (trCBD-LCD, GNATD-only and regulatory domain-only (RD-only)) were properly folded as evidenced 268 by size exclusion chromatography (Figure 4a). As expected from our structural data, the RD -only 269 mutant formed a tetramer, whereas the trCBD -LCD and GNATD-only mutants existed as monomers 270 based on their elution profiles. As anticipated, the RD -only mutant without catalytic module abolished 271 enzymatic activity. Interestingly, both the GNATD-only mutant and the domain-wise truncation mutants 272 of the regulatory domains also lost their acetyltransferase activity towards Acs (Figure 4b). It is 273 noteworthy that the trAGD mutant, which showed aggregation, likely experienced a loss of activity 274 related to the instability of its truncated structure (Extended Data Figure 4a). The loss of enzymatic 275 activity in both the GNATD -only and truncated regulatory domain mutants suggests the necessity of 276 oligomeric conformation and highlights the critical interplay between these domains for the 277 acetyltransferase function of PatZ. 278 279 To investigate whether and how AcCoA binding to the regulatory domain affects PatZ activity, we 280 analyzed the AcCoA binding affinity of PatZ using isothermal titration calorimetry (ITC). As anticipated 281 from a previous study on S. enterica Pat31 and our structural data in this study (Figures 2 and 3), AcCoA 282 binding to wild-type PatZ exhibited a biphasic curve, best fitting to a two-site binding model. One binding 283 site demonstrated a much stronger affinity (K d1 = 0.60 μM) compared to the other (K d2 = 3.98 μM) 284 (Figure 4c). However, when residues R26 and R81 within the AcCoA-binding pocket of PatZ (see Figure 285 2d) was mutated to Glu, disabling AcCoA binding to the RD, the resulting RR mutant of PatZ showed 286 one-site binding (Kd = 12.10 μM), similar to, but with lower affinity than, the RD -only form (Kd = 5.15 287 μM). These kinetic data suggest that the RD has a higher AcCoA binding affinity than the GNATD and 288 that binding of AcCoA to one site increases the affinity of the other site, indicating positive cooperativity. 289 Notably, the monomeric GNATD -only form exhibited a binding affinity (K d = 8.13 μM) similar to the 290 tetrameric RR mutant, indicating that the AcCoA binding affinity of GNATD is not significantly affected 291 by oligomerization. 292 293 Next, we conducted the PatZ activity assay by measuring the free sulfhydryl group of CoA released 294 after the acetyltransferase reaction. The formation of 2-nitro-5-thiobenzoate (TNB2-) from the reaction 295 between the free sulfhydryl group and DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) was recorded at 412 296 nm. Consistent with previous findings for ecPatZ32 (Hill coefficient = 7.91) and sePatZ31 (Hill coefficient 297 = 2.2), wild -type PatZ exhibited a sigmoidal activity curve, indicative of positive cooperativity (Hill 298 coefficient = 2.21; Khalf = 4.20 μM) (Figure 4d). However, the RR mutant exhibited a typical Michaelis-299 Menten curve and had a significantly lower AcCoA affinity (Km = 11.78 μM) compared to wild-type PatZ. 300 These results confirm that AcCoA binding to the regulatory domain not only increases GNATD's affinity 301 for AcCoA but is also essential for the proper allosteric regulation of PatZ's enzymatic activity. 302 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 303 To understand the mechanistic relationship with AcCoA-binding between the regulatory domains and 304 GNATD, we analyzed the domain-wise structural dynamics of PatZ upon ligand binding using single 305 protomer heterogeneity from our cryoEM images. PatZ displayed varying degrees of structural 306 dynamics with and without AcCoA (Figure 4e, Supplementary Video 4). Specifically, apo-PatZ exhibited 307 significantly higher structural heterogeneity compared to the ligand-bound form, as also evidenced by 308 resolution improvement from 2.56 Å to 1.99 Å. The AGD's motion was less correlated and continuous 309 regardless of AcCoA binding. However, we identified three distinct classes (class I-III, Extended Data 310 Figure 4b) of domain interfaces of the LCD with ~15 Å RMSD in apo -PatZ, while the AcCoA -bound 311 structure showed a well-converged single conformation (Figure 4f). These structural changes in apo -312 PatZ resulted in a relaxed axial intersubunit space between the LCD of one subunit and the CBD of the 313 neighboring subunit. To characterize changes in the interface, we measured the distance between S156 314 and R343 of the neighboring protomer, forming part of the ligand binding pocket (Figure 4g, Extended 315 Data Figure 4c). In the apo -state, this distance expanded to 16.9 Å. In the ligand bound form, the 316 distance converged to 11.9 Å. Notably, the class III map in apo -PatZ displayed attributable density to 317 phosphate with an RMSD of 1.2 Å compared to the AcCoA-bound conformation. Therefore, our analysis 318 indicates that AcCoA binding in the CBD contributes to stabilizing the complex and enforcing a stably 319 closed intersubunit conformation. 320 321 Interestingly, the GNATD itself displays well-defined static structures in both the apo- and ligand-bound 322 states. However, binding of AcCoA to PatZ induces significant domain -wise movement in GNATD, 323 specifically a ~37° rotation and a ~40° tilt relative to the regulatory domain from the apo - to AcCoA-324 bound conformation (Figure 4h, Supplementary Video 4). We identified that the regulatory domains 325 alter their specific interactions with the GNATD upon ligand-induced conformational transition. In apo-326 PatZ, the N-terminal end of CBD (Figure 4i) interacts with the β-sheet of gating motif, anchoring GNATD 327 in a down-position. Conversely, in the ligand-bound conformation, the AGD and the interdomain loop 328 (E451-S561) establish new specific interactions with the hinge region (E736, Q772, N769) of GNATD, 329 stabilizing GNATD in an up-position (Figure 4h). 330 331 Importantly, in the apo -PatZ, the tethered configuration limits the space between GNATD and CBD, 332 resulting in the closure of the substrate access channel, with the β -sheet of gating motif physically 333 obstructing the interaction between donor AcCoA and the substrate (Figure 4j, left panel). Binding of 334 AcCoA to both the CBD and GNATD opens the substrate access channel and triggers the 335 rearrangement of the gating motif into an active conformation (Figure 4j, right panel). Taken together, 336 the regulatory domain is essential for the activation of GNATD, and AcCoA binding to both the CBD 337 and GNATD positively cooperates in allosteric regulation to facilitate PatZ's enzymatic reaction, which 338 is in accordance with the ITC and DTNB results (Figure 4c,d). Notably, considering that the close 339 conformation of the regulatory domain (class III in apo-PatZ) still displays a down-position of GNATD, 340 it is logical to conclude that AcCoA binding to GNATD and the switching interaction from the regulatory 341 domain are both prerequisite events for PatZ activation. 342 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 343 Regulatory domain directly shapes Acs binding pocket, enhancing substrate interaction. 344 Upon elucidating a cooperative mechanism between the GNATD and regulatory domains of PatZ for 345 ligand-induced substrate gating, we extended our investigation to explore whether the regulatory 346 domain directly influences binding or specificity of a protein substrate. AlphaFold41 predicted the reliable 347 models showing monomeric interaction within the PatZ-Acs complex structure (Extended Data Figure 348 5). These predictions illustrated that Acs interacts with PatZ and notably, the Acs K609 residue, a known 349 acetylation site, is oriented towards GNATD (Figure 5a) and is positioned within the putative acceptor 350 lysine binding pocket, which is consistent with our experimental analysis (Figure 5a,b). This intricate 351 interaction of Acs is established by an orchestration with the CBD, LCD and GNATD of PatZ. 352 353 Significantly, the predicted PatZ -Acs structure underscores that the regulatory domain forms a 354 distinctive groove and binding surface, accommodating the C -terminal domain of Acs and fostering 355 highly complementary polar interactions (Figure 5b). These specific residues exhibit robust coupled 356 variance, indicative of residue-level coevolution between Acs and the regulatory domain of PatZ. This 357 coevolution suggests a finely tuned evolutionary adaptation, underscoring the additional importance of 358 the regulatory domain for PatZ's functional integrity. 359 360 To validate the impact of regulatory domain and Acs interaction on enzyme activity, we identified and 361 mutated coevolved residues at the interface between Acs-CTD and the regulatory domain of PatZ. We 362 engineered R395A/Y398A and N227A/K228A mutants of PatZ (Figure 5c) and evaluated their enzyme 363 activity compared to the wild-type over time. Our results disclosed a pronounced decrease in enzyme 364 activity for both mutants (Figure 5d), signifying the critical role these residues play in maintaining 365 enzymatic function. This significant reduction in activity underscores the importance of the regulatory 366 domain in facilitating proper substrate binding and, consequently, effective catalysis. 367 368 369

370 371 We characterized multiple ligand -PatZ interactions and demonstrated that ligand -induced 372 conformational changes facilitate the allosteric regulation of the catalytic activity (Figure 4, 6a). Our 373 investigation delineates PatZ’s working mechanism and highlights key aspects of how ligand binding 374 influences PatZ activity and regulation (Figure 6a). 375 376 Gating motif contributes to auto-regulation of GNATs 377 Located near the substrate binding site in GNATD, the gating motif (V746-T763) undergoes significant 378 structural changes, demonstrating the mechanism of opening and closing a passage that connects the 379 donor AcCoA and acceptor protein substrate (Figure 4h-j, 5). In the absence of AcCoA, the gating motif 380 sterically occludes substrate binding sites and masks the passage, indicating inhibited conformation 381 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint (Figure 4h-j). Upon AcCoA binding, the gating motif coordinates with the AcCoA to expose the substrate 382 binding sites and aligns the proximity between AcCoA and protein substrates, indicating activated 383 conformation (Figure 4h -j). AcCoA induced -structural changes in GNATs have been previously 384 discussed in the studies of RimL from S. typhimurium42, Rv0819 from M. tuberculosis43 and ssPat from 385 Sulfolobus solfataricus44. RimL and ssPat are GNATD only enzymes without regulatory domain (Type 386 IV GNAT), while Rv0819 is a tandem-repeated GNAT enzyme (Type V GNATs). In these studies, the 387 homologous region with a gating motif is referred to as α1 -α2 loop, mobile loop or bent helix, which 388 remain unresolved in the absence of AcCoA due to their intrinsic dynamic features. This loop becomes 389 rigidly ordered by AcCoA binding, resulting in the formation of a substrate binding pocket, similar to the 390 gating motif of PatZ. Notably, the mutation study of ssPat has shown that the bent helix is essential for 391 enzyme activity. 392 393 Importantly, this structural component, the gating motif in GNATs, is not only present in prokaryotes but 394 also preserved in eukaryotes, including humans (Extended Data Figure 7), which suggests a common 395 mechanism of GNATs’ auto-regulation across species. Collectively, based on the clear visualization of 396 the inhibitory and active conformations of GNATD in PatZ, we propose an auto-regulatory mechanism 397 of GNATs mediated by the gating motif. Interestingly, despite the high structural similarity of GNATs, 398 the gating motif exhibits relatively low sequence similarity45 (Extended Data Figure 7). Since the gating 399 motif also contributes to the formation of the substrate binding pocket, it is tempting to speculate that 400 the gating motif may be involved in substrate specificity, which requires further studies. 401 402 Regulatory domain interaction mechanistically regulates PatZ activity 403 We characterized that ligand binding to the regulatory domain significantly stabilizes intersubunit 404 interface and rigidifies PatZ's homotetrameric architecture (Figure 4g). Concurrently, this 405 conformational stabilization of the regulatory domain is correlated with the activation process in the 406 gating motif induced by AcCoA binding to GNATD (Figure 4i). These cooperative structural events result 407 in the domain-wise conformations of the up- and down-positioning of the GNATD. The gating motif of 408 GNATD interacts with the CBD in the absence of AcCoA, leading to the down -positioning of GNATD 409 (Figure 4i, left panel). In this down position, the gating motif tethered by the regulatory domain illustrates 410 a restricted space for the substrate access channel (Figure 4j, left panel), indicating an inhibitory 411 conformation. Upon AcCoA binding, the gating motif loses its interaction with CBD, allowing GNATD to 412 refresh its interaction with AGD and adopt an up-positioned conformation (Figure 4i, right panel). This 413 position creates a widely exposed space between GNATD and CBD, allowing protein substrates access 414 to the substrate binding site (Figure 4j, right panel), indicating an activated conformation. 415 416 Interestingly, the stabilized structure with phosphate bound PatZ (Extended Data Figure 4b,c) displays 417 the down position of GNATD and the inactive conformation of the gating motif. The regulatory domain 418 of this structure is identical to that of the AcCoA -bound structure, implying that the stability of the 419 regulatory domain alone cannot transform GNATD into its active conformation. GNATD alone does not 420 also have enzymatic activity and thus suggest that AcCoA binding to both CBD and GNATD is a 421 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint prerequisite for activating PatZ. Importantly, we (Figure 4c,d) and others 31 have demonstrated the 422 cooperative relationship of AcCoA binding between GNATD and CBD. AcCoA binding to the wild-type 423 GNATD is approximately 2 times higher than to the GNATD -only protein and 6.6 times higher to the 424 regulatory domain than to GNATD (Figure 4c). These findings collectively suggest that AcCoA binding 425 to CBD reduces the dynamics of the regulatory domain, enhances the AcCoA binding to GNATD, 426 facilitates the rearrangement of the gating motif, and activates the enzyme. 427 428 Therefore, our study elucidates the active and inactive conformations in the cooperation between 429 GNATD and the regulatory domain and highlights PatZ’s activity regulation mechanism. This elegant 430 mechanism employs a dual strategy that switches between active and inactive conformations and 431 allosterically modulates enzyme activity upon cooperative AcCoA binding. 432 433 Regulatory domain contributes PatZ’s substrate specificity 434 Prior studies have highlighted the unique specificity of PatZ for Acs32,46, and it has been also observed 435 that strains lacking PatZ do not produce acetylated Acs 47. Through our observations, we identified a 436 structural opening event in GNATD, in cooperation with CBD, that forms an Acs-specific binding pocket 437 (Figure 5). These findings validate additional functional layers of the regulatory domain. Moreover, it is 438 evident that GNATD alone in PatZ lacks enzymatic activity toward Acs 40 (Figure 4b), and point 439 mutations in the regulatory domain that disrupt Acs interaction significantly reduce Acs acetylation 440 (Figure 5c,d). Our results further demonstrate that Acs binds complementary to the substrate binding 441 site formed by both GNATD and the regulatory domain (Figure 5a,b). This is supported by the 442 observation that E. coli-derived YiaC, which lacks a regulatory domain (GNAT type IV), is inactive to 443 Acs (Supplementary Data Figure 8). The residues involved in Acs-PatZ interaction are highly conserved, 444 suggesting a finely tuned evolutionary adaptation that underscores the regulatory domain's critical 445 importance for PatZ's function concerning Acs. 446 447 While we elucidate substrate-specific mechanisms, it is noteworthy that PatZ is recognized for its ability 448 to acetylate a diverse array of protein substrates within the bacterial proteome, with an estimated around 449 1000 substrates compared to around 20~600 for GNATD -only enzymes like YjaB, YiaC, RimI and 450 PhnO48. Although these acetyl -substrate repertoires overlap significantly, PatZ exhibits a unique 451 capability for the specific targeting of substrates32,46. 452 453 Our study implies that PatZ employs dual modes of substrate recognition: a general acetyltransferase 454 function for exposed lysines across a broad spectrum of proteins and a specific mechanism through 455 cooperative substrate recognition involving both GNATD and the regulatory domains (Figure 6a). This 456 dual functionality highlights the sophisticated regulatory mechanisms that enable PatZ to achieve both 457 substrate specificity and enzymatic versatility. 458 459 Diverse roles of regulatory domains in GNATs family for bacterial physiology 460 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Protein acetylation plays crucial roles in cellular physiology by impacting various processes and 461 regulatory mechanisms49. Present in all living organisms across the Bacteria, Archaea, and Eukarya 462 domains50, protein acetyltransferases, including GNATs suggest that protein acetylation has existed 463 since the earliest stages of life, predating the Last Universal Common Ancestor (LUCA) more than 4 464 billion years ago51. Despite the vast evolutionary divergence from LUCA, essential cellular functions 465 such as genetic information processing and core metabolic pathways have been conserved across 466 phylogeny52. 467 468 Our study reveals that the mechanistic coupling of AcCoA binding to the regulatory domain modulates 469 the activity of the Type I GNATs, such as PatZ. Given that unique types of regulatory domains in other 470 GNAT’s can bind different ligands, this variation suggests that bacteria regulate GNATs’ activity in 471 response to diverse metabolic conditions (Figure 6b). We conducted a protein structure-based 472 phylogenetic analysis against the AlphaFold Protein Structure Database53 to explore the distribution of 473 GNAT types in bacteria (Extended Data Figure 6). Notably, we found that while bacteria can 474 simultaneously possess multiple types of GNATs, Type IV GNATs, which consist solely of the GNATD, 475 exhibit a ubiquitous distribution across the bacterial domain (Extended Data Figure 6e ,f). In contrast, 476 we identified a distinct pattern where other types of GNATs with unique regulatory domains are 477 predominantly found within specific phyla (Extended Data Figures 6e,f). 478 479 Type IV GNATs, processing a single catalytic GNAT domain, are ubiquitously present in bacteria 480 (Extended Data Figure 6e,f), where they regulate nucleoid organization 54, transcription55, and central 481 metabolic pathways56. This ubiquity implies GNATs’ involvement in regulating housekeeping processes. 482 In contrast, Type I, II, and III GNATs possess additional regulatory domains (Figure 6b, Extended Data 483 Figure 6b-d), likely enabling them to sense various environmental conditions and tightly regulate genetic 484 information flow and metabolic fluxes in response to nutrient availability and cellular energy status. 485 486 For instance, in E. coli, the Type I GNAT PatZ uses AcCoA not only as an acetyl -donating substrate 487 but also as a ligand binding to its regulatory domain (Figure 2), thus allosterically regulating its GNAT 488 activity (Figure 4). One of the most studied Type II GNATs is PatA from S. lividans57. While the ligands 489 binding to its regulatory domain are not well understood, predictions using AlphaFold41 and AlphaFill58 490 revealed that AcCoA and ATP bind to PatA’s regulatory domain (Figure 6b). Previous studies have 491 reported that Type III GNATs have their activity regulated by the binding of amino acids, cAMP, and 492 NADP+ to their regulatory domains59–61. Although different types of GNATs adopted various ligands as 493 regulatory cues, these ligands generally represent nutritional and cellular energy status62. 494 495 Interestingly, the activity of Acs is regulated by acetylation via GNATs in most, if not all, bacterial species 496 studied so far4. AcCoA is a crucial metabolite in central metabolism, acting as a crossroad in pathways 497 such as glycolysis, gluconeogenesis, the tricarboxylic acid cycle, the glyoxylate bypass, lipid 498 metabolism, and amino acid synthesis63. Nearly all life forms have evolved to adopt AcCoA as a key 499 metabolic intermediate, making it essential to maintain its levels for supporting all cellular processes. 500 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint While different bacterial phyla have adopted different types of structures of regulatory domains (Figure 501 6b-d), a common function of GNATs appears to be the regulation of housekeeping processes by 502 sensing diverse cellular cues. 503 504 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint

505 506 Constructs design and molecular cloning 507 The full-length or truncated PatZ gene (UniProt ID: P76594) was cloned into the pETDuet -1 vector 508 (Novagen) with a 6xHis tag at the C-terminus. The full-length Acs gene (UniProt ID: P27550) was cloned 509 into the pETDuet-1 vector with an N -terminal 6xHis tag. All mutants were generated by site -directed 510 mutagenesis. 511 512 Protein expression and purification 513 The recombinant DNA is transformed into the patz, pta deletion ER2566 strain. The transformed cells 514 were grown in LB medium at 37 °C until the O.D 600 value reached 0.6~0.8. The temperature was then 515 lowered to 17 °C, and 1 mM IPTG was added to induce protein expression. After 16 hours of induction, 516 the cells were harvested by centrifugation and the medium was completely discarded for storage at -517 80 °C. The harvested cells were disrupted using Emulsiflex C3 (Avestin) and the cell debris was 518 removed by centrifugation at 20,784 g for 30 min. The supernatant is filtered before purification by FPLC. 519 And the filtered samples were purified by Ni-NTA affinity chromatography using Histrap HP pre-packed 520 columns (Cytiva). After washing with wash buffer (25 mM Tris -HCl pH 7.4, 500 mM NaCl, 20 mM 521 imidazole, 1 mM MgCl 2, 10% glycerol) elution was performed with elution buffer (20 mM HEPES pH 522 7.4, 500 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT) with 300 mM Imidazole. The purified protein 523 samples were then further purified by size exclusion chromatography (SEC) using a Superose 6 524 Increase column (Cytiva) in a buffer containing PBS with additional 100 mM NaCl. Acs was cultured in 525 TB medium under the same conditions. The purification procedure followed the same protocol: After 526 extensive washing with wash buffer (25 mM Tris -HCl pH 7.4, 500 mM NaCl, 40 mM imidazole, 10% 527 glycerol), elution was carried out using elution buffer (25 mM Tris-HCl pH 7.4, 500 mM NaCl, 300 mM 528 imidazole, 10% glycerol). and then further purified by SEC using a HiLoad 16/600 superose 6 pg (Cytiva) 529 in a buffer containing 25 mM Tris-HCl pH 7.4, 500 mM NaCl, 5% glycerol. 530 531 CryoEM specimen preparation and imaging condition 532 Purified protein sample was adjusted to 1.5 mg/mL and vitrification was conducted using the Vitrobot 533 system (ThermoFisher). 3 μL samples were applied to the discharged UltrAuFoil R1.2/1.3 gold 300 534 mesh (Quantifoil). Grids were blotted for 3 sec and plunged into liquid ethane at 100% humidity at 8 °C. 535 The grids were roughly screened on a Glacios operated at 200 keV (cryoTEM, ThermoFisher) and 536 equipped with a Falcon 4 direct electron detector. The nicely vitrified grids were placed on a 300 keV 537 Titan Krios G4 (ThermoFisher) equipped with a Falcon 4i direct electron detector. Data was acquired 538 in EER format at a pixel size of 0.723 Å/pixel using aberration-free image shift (AFIS) to accelerate the 539 data acquisition. and total exposure dose was 50 e/A2 with -0.8 to -1.5 μm defocus range. More details 540 about imaging conditions are described in Supplementary Table 1. 541 542 CryoEM image processing 543 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint The EM data processing workflow for all datasets is illustrated in Extended Data Figures 1 and 2. The 544 processing was conducted using CryoSPARC v4.4.1 packages, and a comparable strategy was 545 employed for all datasets. Firstly, the beam -induced motion of the movies was corrected using Patch 546 motion correction, and the contrast transfer function (CTF) of each micrograph was calculated using 547 Patch CTF estimation. Some micrographs were excluded due to insufficient resolution of the CTF and 548 defocus range. Subsequently, about 3,000 particles were selected via blob-based picking from a subset 549 of 30 micrographs, resulting in the generation of 2D templates. Finally, a considerable number of 550 particles were automatically selected from the entirety of the micrographs using the templates. The 551 extraction of particles was conducted using a box size of 448 X 448 pixels. Subsequently, several 552 iterations of 2D classification were performed, and 3D volumes were generated without alignment 553 through Ab -initio reconstruction. The optimal volume class with D2 symmetry was subjected to 554 Homogeneous 3D refinement. To enhance the overall quality of the particles and volumes, Topaz 555 particle picking and Reference-based motion correction (beta) were applied based on the initially refined 556 map. Subsequently, Final maps were generated, followed by global and local CTF correction to 557 enhance the map. 558 559 For asymmetric unit analysis, each particle was expanded according to D2 symmetry. The optimal 560 volumes for each domain and subunit were classified through multiple rounds of 3D classification and 561 subjected to local refinement using the corresponding masks without any symmetry enforcement. 562 These refinements significantly improved domain resolution and features. Structural heterogeneity was 563 characterized by iterative 3D classification and 3D variability analysis. To generate representative final 564 maps, locally refined maps were aligned into the consensus refined map and patched using the “vop 565 maximum” command in UCSF ChimeraX 64 . These focused refinement maps and the merged maps 566 were subsequently used for model building, refinement, and analysis. 567 568 Model building and refinement 569 Initial models were generated using AlphaFold and subsequently fitted to the map using ISOLDE. 570 Manual model fitting was then performed using the Coot program, followed by model refinement using 571 Real-space refinement in Phenix. The refinement results and model quality were numerically assessed 572 through Real-space refinement output values and MolProbity scores. The structure of liganded -PatZ 573 was built using the structure obtained from apo-PatZ as a template, and model refinement was carried 574 out following the same methodology as for apo-PatZ. 575 576 X-ray crystallography, data processing and refinement 577 AGD of PatZ (T463 -S695) was subcloned into pET28a and overexpression and purification were 578 conducted using the same method but with an SEC buffer containing 20 mM HEPES -NaOH pH 7.5, 579 150 mM NaCl, 1 mM MgCl2, 1 mM DTT. Concentration of purified samples was adjusted to 10 mg/mL 580 and co-crystallized with 5 mM ATP. Initial crystallization screening was conducted by using a Mosquito 581 robot (TTP Labtech) and a single, appropriate size of crystals appeared at 0.2 M potassium sodium 582 tartrate, 20 % (w/v) PEG 3350. 20 % ethylene glycol was added as a cryo-protectant and flash frozen 583 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint in liquid nitrogen. Crystal diffraction was carried out at the Pohang accelerator laboratory 5C (PAL-5C) 584 beamline (wavelength= 0.97942 Å)65 on 100K temperature. Diffraction data sets were processed and 585 scaled with the programs HKL2000 66 and imosfilm 67 . The phasing information was solved by a 586 molecular replacement (MR) method using the cryoEM structure. MOLREP68, REFMAC569 , and COOT 587 were used for MR, structure refinement and further modeling, respectively. Figures were prepared using 588 PYMOL and ChimeraX. The Ramachandran plot analysis of the final model revealed that 96.44% of 589 residues were in favored regions, 3.34% in allowed regions, and 0.22% in disallowed regions. 590 591 In vitro acetylation assay: Western blot 592 Acs (1.3 μM) was incubated with wild-type or mutant PatZ (425 nM) in the presence of 1 mM AcCoA 593 (Cayman) or 1-10 mM of AcP (Merck) in an assay buffer (25 mM sodium phosphate pH 8.0, 0.5 mM 594 EDTA, 100 mM NaCl) at 37 °C for 10 min, if not otherwise specified. Proteins were then separated by 595 4-20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) and 596 transferred onto a nitrocellulose membrane in transfer buffer for 70 min at 200 mA at 4°C. After transfer, 597 membranes were blocked with 4.5% BSA in PBS-T (4 g NaCl, 100 mg KCl, 720 mg Na2HPO4, 123 mg 598 KH2PO4, 250 μl Tween -20 in 500 ml) for 60 min at room temperature. After blocking, the membrane 599 was washed using PBS-T once and then incubated overnight at 4°C with the first antibody, Anti-acetyl 600 lysine antibody (ICP0380, Immunechem), diluted 1:5000 in PBS -T containing 4.5% BSA. After the 601 treatment with the first antibody, the membrane was washed three times with PBS -T for 10 minutes 602 each. Then, it was incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP)linked secondary 603 antibody (LF-SA8002, Abfrontier), diluted 1:5000 in PBS -T containing 4.5% BSA, for 60 min. The 604 membrane was washed 4 times with PBS-T for 10 min each time, incubated in Western HRP substrate 605 (WBLUF0100, Millipore), and imaged in the Luminograph II (ATTO)70. 606 607 In vitro acetylation assay: DTNB assay 608 The reaction mixture was prepared by adding wild -type or mutant PatZ (1 μM), Acs protein (20 μM), 609 DTNB (100 μM, 5,5’-dithio-bis-(2-Nitrobenzoic Acid, Thermo), and AcCoA (from 0 to 20 μM, Cayman) 610 in a 200 μL volume of reaction buffer (25 mM sodium phosphate, pH 8.0, 0.5 mM EDTA, 100 mM NaCl). 611 Given that both Acs and AcCoA serve as substrates for PatZ, experiments utilized lower concentrations 612 of AcCoA compared to Acs to obtain an accurate initial velocity of CoA release. Absorbance at 412 nm 613 wavelength was monitored every 5 seconds for 10 minutes at 37°C using a Flex3 microplate reader 614 (Molecular Devices). A standard curve was created using CoA (Avanti) concentrations ranging from 0 615 to 100 μM. Velocity was determined by measuring the consumption of AcCoA using the absorbance 616 at 412 nm and then converting this value using the CoA standard curve. This consumption rate was 617 then divided by the time (in minutes) and the amount of PatZs in mg. Velocity and AcCoA concentration 618 were used to fit the wild -type PatZ to a sigmoidal allosteric curve, while the mutants were fitted to a 619 Michaelis-Menten curve, using Prism (GraphPad) analytical software, as used in a previous study31. 620 621 Isothermal Titration Calorimetry (ITC) assay 622 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint The protein sample was prepared in the ITC buffer (1X PBS, 100 mM NaCl), and AcCoA was prepared 623 in the same ITC buffer at a 15 -fold concentration of each protein. For titration, samples were loaded 624 into a 96-deep-well plate three consecutive wells filled for a single titration: samples for the reaction 625 cell, the injection syringe, and the binding buffer for pre -rinsing the reaction cell. All titrations were 626 performed using the automated MicroCal AutoPEAQ ITC instrument (Malvern Panalytical, 627 Worcestershire, UK) with 40 injections at 25°C. The experimental data were analyzed using MicroCal 628 PEAQ-ITC analysis software with a single-site/double-site binding model. 629 630 Phylogenetic analysis 631 We downloaded six predicted structures representing each type of GNAT acetyltransferase from the 632 AlphaFold Protein Structure Database53 (AFDB) May 2024 release: P76594, A0A0M8Z1A2, O05581, 633 D9SZU3, Q1D7F7 and P0A944 for type I, type II, cAMP-binding type III, ACT type III, NADP+-binding 634 type III and type IV, respectively. Each structure was searched against AFDB50 (AFDB clustered by 635 50% 3Di sequence identity) using Foldseek71(version a2f62e) to obtain alignments with at least 90% of 636 both 3Di sequences are covered (foldseek search -c 0.9 --cluster-search 1 --max-seqs 100000). 637 Resulting alignments were then converted into Kraken2 sample report format and visualized as Sankey 638 diagrams with Pavian72(version 1.0). 639 640 We collected whole genome assemblies of 300 bacterial species from NCBI GenBank 2024 update73, 641 including Escherichia coli , Streptomyces lividans , Micromonospora aurantiaca , Mycobacterium 642 tuberculosis, Myxococcus xanthus and 295 species randomly sampled across the bacterial domain. 643 Four archaeal species under genus Methanococcus were included as an outgroup. Maximum likelihood 644 tree was then inferred from the concatenation of amino acid alignments of 81 bacterial core genes 645 extracted from the assemblies with UBCG74 (version 2.0), applying JTT+F+I+G evolutionary model of 646 IQ-TREE75(version 2.2.2.7). 647 648 Finally, we’ve put the representative acetyltransferases predicted/experimental structures which has 649 different types of regulatory domains: Streptomyces lividans PatA (slPatA, AIJ12884.1) for Type II and 650 Escherichia coli YiaC (ecYiaC, UniProt: P37664), while Type III includes Mycobacterium tuberculosis 651 Pat (MtPat, PDB ID: 4AVB), Micromonospora aurantiaca PatB ( maPatB, UniProt: D9SZU3) and 652 Myxococcus xanthus ActC (mxActC, UniProt: Q1D7F7). slPatA displays similarities to those of Type I 653 with ligands, but the three of Type III GNATs bind to highly specific ligands, including cAMP, Cys/Arg 654 and NADP +. Upon cAMP and Cys/Arg, mtPat and maPatB efficiently acetylate their substrates, 655 respectively. But interestingly, mxActC is inhibited when NADP+ is bound. 656 657 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint DATA, MATERIALS, AND SOFTWARE AVAILABILITY 658 659 Atomic coordinates of the apo PatZ, liganded PatZ and ATP bound AGD of PatZ models were deposited 660 in the RCSB PDB under accession numbers 9SIQ, 9IT0 and 9ISB, respectively. The cryoEM maps 661 were deposited in the EMDB under accession numbers EMD -60849 (apo PatZ) and EMD -60853 662 (liganded PatZ). All code used for cryoEM data analysis, structure determination and refinement are 663 publicly available. 664 665 666

667 668 This work has been supported by the Korean National Research Foundation (2019R1C1C1004598, 669 2020R1A5A1018081, 2021M3A9I4021220, 2019M3E5D6063871, 2020R1A6C101A183) and SUHF 670 foundation to S.-H.R and NRF -2018R1A5A1025077, NRF-2019R1A2C2004143, RS-2024-00335765 671 to Y.-J. S. The cryoEM data was collected and processed at The Center for Macromolecular and Cell 672 Imaging (CMCI), Institute for Basic Science (IBS), Global Science experimental Data hub Center 673 (GSDC) at Korea Institute of Science facilities. We thank members of CMCI for their discussions and 674 suggestions. We thank members of the Frydman lab for useful discussions and suggestions. We thank 675 the staff scientists for assistance at the beamline 5C of Pohang Light Source. 676 677 678 AUTHOR INFORMATION 679 680 School of Biological Sciences, Institute of Molecular Biology and Genetics, Seoul National University, 681 Seoul, 08826, Republic of Korea 682 Jun Bae Park, Yu-Yeon Han & Soung-Hun Roh 683 684 School of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, 08826, 685 Republic of Korea 686 Gwanwoo Lee, Kyoo Heo & Yeong-Jae Seok 687 688 Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, 08826, Republic of Korea 689 Dongwook Kim 690 691 Interdisciplinary Program in Bioinformatics, School of Biological Sciences, Institute of Molecular Biology 692 and Genetics, Artificial Intelligence Institute, Seoul National University, Seoul, 08826, Republic of Korea 693 Martin Steinegger 694 695 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, 50 696 Yonsei-ro, Seoul, 03722, Republic of Korea 697 Hyun-Soo Cho 698 699 Department of Chemistry, Chonnam National University, Gwangju, 61186, Republic of Korea 700 Hyosuk Yun, Chul Won Lee 701 702 School of Biological Sciences and Institute of Microbiology, Center for RNA Research, Institute for Basic 703 Science, Seoul 08826, Republic of Korea 704 Jeesoo Kim, Juhee Park & Jong-Seo Kim 705 706 Author Contributions 707 S.-H.R. and Y.-J.S. conceived the project. J.B.P. and G.L. lead the project. J.B.P. and G.L. designed 708 the experiment. J.B.P., G.L., Y .-Y.H. and K.H. performed molecular cloning, protein expression and 709 purification of the proteins. CryoEM sample preparation, data collection and data processing were 710 carried out by S .-H.R., J.B.P. and Y .-Y.H.. S.-H.R., J.B.P. and Y .-Y.H. analyzed the data, built and 711 refined the model. For the X -ray crystallography, J.B.P. performed molecular cloning, protein 712 expression, protein purification, crystallization, X -ray diffraction, data processing, model building and 713 refinement under guidance of H .-S.C.. Y.-J.S. and G.L. conducted the enzymatic activity assay and 714 biochemical assay. M.S. and D.K. performed phylogenetic analysis. All authors analyzed and discussed 715 the results. J.B.P., G.L., Y.-Y.H. and D.K. wrote the paper with help from all authors. 716 717 Competing interests 718 The authors declare no competing interest. 719 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint

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It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 42, 243–246 (2024). 879 72. Breitwieser, F. P. & Salzberg, S. L. Pavian: interactive analysis of metagenomics data for 880 microbiome studies and pathogen identification. Bioinformatics 36, 1303–1304 (2020). 881 73. Sayers, E. W. et al. GenBank 2024 Update. Nucleic Acids Res. 52, D134–D137 (2024). 882 74. Kim, J., Na, S.-I., Kim, D. & Chun, J. UBCG2: Up-to-date bacterial core genes and pipeline for 883 phylogenomic analysis. J. Microbiol. 59, 609–615 (2021). 884 75. Minh, B. Q. et al. IQ-TREE 2: New Models and Efficient Methods for Phylogenetic Inference in 885 the Genomic Era. Mol. Biol. Evol. 37, 1530–1534 (2020). 886 887 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint FIGURES and LEGENDS 888 889 890 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 891 Figure 1. Structure of the apo-PatZ. 892 a, Domain architecture of bacterial Nε -lysine acetyltransferase that includes the GNAT domain. b, 893 Comparison of enzymatic acetylation by PatZ and nonenzymatic acetylation by acetyl -phosphate. 894 Western blot analysis using anti-acetylated lysine antibody. CBB denotes coomassie brilliant blue. c, 895 Analysis of the tetrameric state and purity of PatZ using size exclusion chromatography and SDS-PAGE. 896 d, CryoEM map and refined model. Each color denotes a distinct subunit. e, The structural arrangement 897 of PatZ, consisting of four subdomains. f, The interaction between subunits or subdomains forming the 898 lateral interface g, and axial interface. 899 900 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 901 902 903 Figure 2. Structure of the liganded-PatZ. 904 A, Ligand bindings to tetrameric PatZ and their binding sites. Four ATPs were docked into the tetramer 905 structure based on the ATP bound AGD crystal structure. B, PatZ subdomain schematic and its ligand 906 binding site. 2Fo-Fc electron density map of ATP and cryoEM maps of two AcCoAs and phosphate. C, 907 Molecular interactions between PatZ GNATD and AcCoA. D, Molecular interactions between the axial 908 dimer interface and phosphate, and between PatZ CBD and AcCoA. E, Interaction between PatZ AGD 909 and ATP. *’w’ denotes water molecule. Hydrogen bonds and electrostatic interactions are represented 910 by black dashed lines. 911 912 913 914 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 915 Figure 3. AcCoA-induced conformational change and catalytic residues in GNATD. 916 A, Structural rearrangement of GNAT (red cartoon). Substrate gating motifs in the apo -state and 917 AcCoA-bound state are illustrated in blue color and purple color, respectively. AcCoA is shown in ball-918 and-stick style. B, Molecular interaction between AcCoA and PatZ GNATD. Magenta color is part of the 919 substrate gating motif of GNATD. C, Structural influence of the AcCoA -induced movement of gating 920 motif on the formation of the substrate binding site. D, Electrostatic surface charge and amino acid 921 residues comprising the acceptor lysine binding site in GNATD. E, Comparison of relative functional 922 level between wild-type and point mutants which comprise acceptor lysine binding pocket. Western blot 923 analysis was performed using an anti-acetylated lysine antibody. F, Homology structure based catalytic 924 mechanism prediction of PatZ using slPatA and seAcs complex structure. Hydrogen bonds are 925 represented by black dashed lines. G, Proposed acetyltransferase mechanism of PatZ. 926 927 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 928 929 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Figure 4. Allosteric coupling of regulatory domain to GNATD. 930 A, Schematic of domain truncation construct design and its size exclusion chromatography peak pattern. 931 ‘tr’ denotes truncation. B, Comparative enzymatic activity analysis of subdomain truncated constructs 932 via western blot. Western blot analysis was performed using an anti-acetylated lysine antibody c, ITC 933 profiles of AcCoA binding to wild -type, truncated forms and RR (R26E/R81E) mutant of PatZ. D, 934 Comparative enzymatic activity analysis of wild-type, RR mutant and GNAT-only constructs of PatZ. E, 935 Structural variability comparison between apo - (left) and liganded -PatZ (right). F, Comparison of 936 backbone RMSD between apo - and liganded-PatZ (purple) and backbone RMSD within apo -PatZ 937 variability (red) and liganded -PatZ variability (blue). Asterisk refers to the substrate gating motif of 938 GNATD. G, Ligand binding-mediated structural change of PatZ regulatory domain h, Structural effect 939 of AcCoA binding on the interaction between GNATD and regulatory domain. Hydrogen bonds are 940 represented by dashed lines. I, Role of substrate gating motif in changes in interaction between GNATD 941 and regulatory domain following AcCoA binding j, Substrate access channel formed by AcCoA -942 mediated GNATD conformational change and the role of substrate gating motif. Magenta color is a 943 space filling model of substrate gating motif. 944 945 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 946 947 948 Figure 5. The regulatory domain of PatZ contributes to the formation of Acs binding pocket. 949 a, Predicted structure of the E.coli PatZ-E.coli Acs complex using AlphaFold 41. Each domain is 950 represented in a different color using cartoon -style representation. Acs K609 is represented in stick 951 style. b, Electrostatic surface potentials of Acs and PatZ. Red and blue for negative and positive charges, 952 respectively, and white color represents the neutral area. The circularly linked different parts represent 953 the interacting components. c, Acs-CTD and PatZ-LCD interaction (left panel) and Acs-CTD and PatZ-954 CBD interaction (right panel). The circled regions denote the positions of the labeled residues. d, Time-955 dependent comparison of PatZ mutant activity relative to wild -type. Western blot analysis using anti -956 acetylated lysine antibody (left panel) and normalized activity comparison graph (right panel). 957 958 959 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 960 961 Figure 6. Phylogenetic analysis and catalytic mechanism 962 a, Schematic illustration of ligand -induced domain-specific conformational changes regulating PatZ 963 catalytic activation. b, Visualization of Type I, Type II, cAMP -binding type III, ACT type III, NADP+ -964 binding Type III and Type IV c, Venn diagram showing the overlap of GNAT types. Each number 965 represents the count of bacterial species containing the corresponding GNAT type. 966 967 968 969 970 971 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint EXTENDED DATA and LEGENDS 972 973 974 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 975 Extended Data Figure 1. CryoEM processing workflow of apo -state PatZ. a, Schematic of data 976 processing pipeline with representative cryogenic electron microscopy micrographs and two -977 dimensional averages. b, Angular distribution of views in the 3D reconstruction (left view and right view, 978 rotated by 90˚). c, Gold-standard fourier shell correlation (GSFSC) curve. d, Map in front view, colored 979 according to local resolution. e, CryoEM density map features corresponding to the four subdomains. 980 f, Side chain Q-score distribution across the four domains. 981 982 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 983 984 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Extended Data Figure 2. CryoEM processing workflow of liganded-PatZ and 2Fo-Fc electron density 985 map of ATP in ATP bound AGD crystal structure. a, Schematic of data processing pipeline with 986 representative cryogenic electron microscopy micrographs and two-dimensional averages. b, Angular 987 distribution of views in the 3D reconstruction (left view and right view, rotated by 90˚). c, Gold-standard 988 fourier shell correlation (GSFSC) curve. d, Map in front view, colored according to local resolution. e, 989 CryoEM density map features corresponding to the four subdomains. f, Side chain Q-score distribution 990 across the four subdomains. g, Model refinement results for two AGD chains located in a single 991 asymmetric unit of the crystal, with 2Fo-Fc electron density map of ATP contoured at 1σ. 992 993 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 994 995 Extended Data Figure 3. Structural similarity between GNATD of ecPatZ and slPatA, and sequence 996 conservation level of PatZ E809. a, Structural similarities between ecPatZ GNATD and slPatA GNATD. 997 b, ConSurf analysis of sequence conservation for PatZ E809. 998 999 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 1000 Extended Data Figure 4. a Comparison of relative functional level between wild-type and truncated 1001 mutants. Asterisk mark refers to the truncated PatZ s. b, Structural comparison of three distinct 1002 conformational states (Classes I -III) of apo -state PatZ LCD identified through 3D classification . c, 1003 Quantitative comparison of conformational changes in the regulatory domain induced by ligand binding. 1004 1005 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 1006 1007 Extended Data Figure 5. Prediction of PatZ-Acs complex structure using AlphaFold3. a, The Predicted 1008 Aligned Error (PAE) matrices calculated via AlphaFold3. b, pLDDT for all residues belonging to the Acs 1009 and PatZ. c, 3D visualization of prediction error estimation using PAE plot focused on PatZ -Acs 1010 interaction part. Stick style refers to residues originating from two chains within a 4 Å distance. The 1011 coloration follows the pLDDT color spectrum. 1012 1013 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 1014 Extended Data Figure 6. The PAE matrices that are calculated via AlphaFold3. a, E.coli YiaC (Type 1015 IV). b, S. lividans PatA (Type II). c, M. aurantiaca PatB (Type III). d, M. xanthus ActC (Type III). e, 1016 Domain-wide distribution of GNAT acetyltransferase types across a phylogenetic tree of 300 bacterial 1017 species. Different types are indicated by dots in the concentric circles, where a filled dot indicates the 1018 species containing a structure similar to the representative structure of each type. For each type, 1019 percentage of the species abundance among the bacterial domain as well as the representative 1020 structure are displayed on the location of corresponding species. f, Distribution of GNAT types across 1021 the bacterial domain displayed as Sankey plots. Shown are the ranks of domain (D) and phylum (P), 1022 with the top ten most abundant groups per rank; numbers indicate the amount of underlying species. 1023 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 1024 1025 1026 Extended Data Figure 7. Structural similarity of gating-motif-containing helices. Comparative analysis 1027 of sequence-structure similarity in GNAT gating motifs across eukaryotes and prokaryotes. Distinct 1028 helices containing the gating motif are differentiated by color. Similarities among the sequences are 1029 given a gray background. 1030 1031 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint 1032 Extended Data Figure 8. Western blot analysis using anti -acetylated lysine antibody for comparison 1033 of acetylation by type I E. coli PatZ and type IV E. coli YiaC. 1034 1035 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Supplementary Table 1. CryoEM data collection, refinement and validation statistics 1036 1037 1038 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Supplementary Table 2. X-ray crystallographic data collection and refinement statistics 1039 1040 1041 Supplementary video 1. Tetramer assembly and subdomain composition of PatZ 1042 1043 Supplementary video 2. Ligands that bind to PatZ and their binding sites 1044 1045 Supplementary video 3. Ligand-mediated conformational change of PatZ GNAT domain 1046 1047 Supplementary video 4. Ligand-mediated global conformational change of PatZ 1048 1049 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint N C Abs (280nm) 0 5 10 15 20 25 (mL) (kDa) 150 100 75 670 158 44 17 (kDa) ca b GNATDLCD 1 451 709 886285 N CAGD Catalytic domainRegulatory domain CBD 90˚ 180˚ 90˚ 1 2 3 4 1 2 3 4 1 2 3 4 CoA-binding domain (CBD) Ligase-CoA domain (LCD) ATP-grasp domain (AGD) GNAT domain (GNATD) N C CBD LCD GNATD AGD CBD LCD GNATD AGD Catalytic domain Regulatory domain 1 2 3 4 1 2 3 4 1 2 Lateral interface Axial interface 2 4 CBD LCD 90˚ CBD d e f g 1 2 2 1 K171 Q169 E408 H409S410 2 4 A53 A53 G56 G56 P43 V44 P43V44 Regulatory domain GNAT domain Domain configuration Type I Type II Type III Type IV Type V Acs Anti-AcLys CBB staining Acs PatZ + + + + - - - - - 1 mM AcCoA - + + + - - - - - 1 mM AcP - - - - + + + + - Acs + + + + + + + + + 0m 10m 1h 16h 0m 10m 1h 16h 16h 10 mM AcP - - - - - - - - + .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint ATP AcCoA AcCoA Phosphate 1 2 3 4 1 2 3 4 CBD LCD AGD GNATD ATP AcCoA ATP AcCoA AcCoA AcCoA AcCoA Phosphate A299 A298 R343 A158 A157 S156 phosphate AcCoA LCD-CBD interface K860 R825 G824 L823 K189G822 L821 w V814 L813 V812 N851 F810 GNATD AcCoA L813 V814 N851 K860 R825 A577 Q574 K523 R579 E584 H676 H531 K532S533 AGD K523Q574 R579 E584 H676 H531 ATP a b c W51 T48 S21 R26 N78 R81 AcCoA K23 CBD R81 N78 R26 K23S21W51 T48 A299 A298 A158 A157 S156 w w R343 d e Regulatory module AcCoA binding AcCoA binding ATP binding phosphate binding GNATDLCD 1 451 709 886285 N AGDCBD Catalytic module .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint Substrate binding site AcCoA binding site apo- state AcCoA- state AcCoA binding D750 Y753 M848 N851 AcCoA AcCoA binding a F756 R754 I846 E809 AcCoA f E809 F810 I846 AcCoA E123 E160 K609 V124 H2O slPatA GNATD seAcs CTD (4U5Y): AcCoA I846 F810 E809 H2O K609 ecPatZ GNATD seAcs CTD (Superposition): g b WB CBB Acs Acs + + + + + + - + + + + + + + + + + +PatZ AcCoA Acs WT WT R754A F756A E809A I846A O - O Glu809 CoA-S O PatZ Lysine-substrate O HO Glu809 PatZ AcLys-substrate CoA-SH CoA-SH AcCoA H H O LysH +.. NH2 H H O AcLys NH O PatZ-GNATD Lys-substrate PatZ-GNATD Lys-substrate d e c Substrate- gating motif .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint a NTD CTD GNATD K609 LCD R395 Y398 LCD CTD E570 K228 N227 CBD CTD D634 WB CBB Ac-CoA Acs + + - + + + + + + - + + + + + + + 10 30 10 30 10 30 PatZ WT PatZ R395A,Y398A PatZ N227A,K228A (min) - 0 0 0 R395A, Y398A N227A, K228A 0 0.5 1.0 1.5 PatZ WT Nomalized activity * ** NTD CTD GNATDK609 LCD CBD PatZAcs CBD AGD PatZ Acs b c d (i) (ii) .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint e h i g j c f d 0 20 40 60 80 100 120 1 0 -1 -2 -3 -4 0 1 2 3 0 -10 -20 -30 DP (μM) Ratio Time (min) WT Kd1 0.60 μM Kd2 3.98 μM 0 20 40 60 80 100 120 Time (min) 0 -10 -20 -30 ΔH (kJ/mol) 1 0 -1 -2 -3 -4 0 1 2 3Ratio RD-only Kd 5.15 μM 0 1 2 3Ratio 1 0 -1 -2 -3 -4 DP (μM) 0 -10 -20 -30 0 20 40 60 80 100 120 R26E, R81E Kd 12.10 μM 0 20 40 60 80 100 120 0 -5 -10 -25 ΔH (kJ/mol) -15 -20 1 0 -1 -2 -3 -4 0 1 2 3Ratio GNATD-only Kd 8.13 μM 0 20 40 60 5 10 15 AcCoA (μM) Velocity (μM/min/mg) 0 WT R26E, R81E GNATD-only Time (min) Time (min) 0 5 10 15 20 25(mL) Abs (280nm) 670 158 44 17 (kDa) WT Void WT trCBD-LCD RD-only GNATD-only 1 451 709 886285 461 AGDLCD GNATN CCBD AGD GNATN C GNATN C AGDLCDN CCBD a trCBD-LCD - - + + + + + - + - + + + + + + + + + + + PatZ AcCoA Acs WB WT WT GNATD-onlyRD-only Acs Acs CBB staining LCD GNATN CCBD trAGD GNATD-only trCBD-LCD trAGD RD-only b Variability in apo-PatZ Variability in liganded-PatZ RMSD RMSD 0 Å 20 Å 0 Å 20 Å RMSD (Å) GNATDLCD AGDCBD 0 10 20 30 0 200 400 600 800 0 5 10 15 *apo- vs liganded-PatZ apo-PatZ liganded-PatZ R343 S156 R343 S156 R343 S156 R343 S156 Phosphate + AcCoA 1 2 3 4 1 21 2 1 21 2 AcCoA AcCoA Overall B-factor = 77.6 Interface area (Ų) = 1002.7 Overall B-factor = 39.0 Interface area (Ų) = 2094.8 Relaxed structure Tense structureRMSD=8.1 Å 37° 40° AcCoA Y753 R197 Y194 E7F756 N-term E736 N769 Q772 T452 R450 C Substrate access AcCoA CBD AGD GNATD GNATD CBD AGD CBD GNATD CBD GNATD AcCoA Y753 Y753 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint CoAAc GNATD AGD CBD LCD Gating- motif Acs Substrate a NADP+ cAMP Cys/Arg AcCoAATP PO- 4 AcCoA ATP Regulatory GNAT Type I Type II Type III Type IV 348 112 67 3,582 14,343 1,543 865 317 7 7 93 Type I Type II Type III Type IV CoA Inhibited conformation Gating motif: closed Substrate access channel : closed Activated conformation Gating motif: open Substrate access channel : open Acetyl-Acs Substrate Substrate access Tethered_down position Up position Gating- motif Substrate access Binding site GNATD AGD CBD LCD b c .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint