{"paper_id":"3ecbec50-4911-4974-b39e-b40ccd70801d","body_text":"Structural basis of the catalytic and allosteric mechanism of bacterial 1 \nacetyltransferase PatZ 2 \n 3 \nJun Bae Park1,2$ Gwanwoo Lee1,3$, Yu -Yeon Han1,2$ Dongwook Kim4, Kyoo Heo1,3, Jeesoo Kim1,5, 4 \nJuhee Park1,5, Hyosuk Yun6, Chul Won Lee6, Hyun-Soo Cho7, Jong-Seo Kim1,5, Martin Steinegger1,2,4,8, 5 \nYeong-Jae Seok1,3* and Soung-Hun Roh1,2* 6 \n 7 \n1School of Biological Sciences, Seoul National University, Seoul, 08826, Republic of Korea 8 \n2Institute of Molecular Biology and Genetics, Seoul National University, Seoul, 08826, Republic of Korea 9 \n3Institute of Microbiology, Seoul National University, Seoul, 08826, Republic of Korea 10 \n4Interdisciplinary Program in Bioinformatics, Seoul National University, Seoul, 08826, Republic of Korea 11 \n5Center for RNA Research, Institute for Basic Science, Seoul 08826, Republic of Korea 12 \n6Department of Chemistry, Chonnam National University, Gwangju, 61186, Republic of Korea 13 \n7Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, 50 14 \nYonsei-ro, Seoul, 03722, Republic of Korea 15 \n8Artificial Intelligence Institute, Seoul National University, Seoul, 08826, Republic of Korea 16 \n 17 \n$These authors contributed equally 18 \n* Corresponding author: Soung-Hun Roh, shroh@snu.ac.kr; Yeong-Jae Seok, yjseok@snu.ac.kr 19 \n 20 \nCLASSIFICATION 21 \nBiological Sciences; Biochemistry  22 \n 23 \nKEYWORDS 24 \nPatZ, YfiQ, Acetyltransferase, Allosterism, CryoEM  25 \n 26 \n  27 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nABSTRACT 28 \n 29 \nGCN5-related N-Acetyltransferases (GNATs) play a crucial role in regulating bacterial metabolism by 30 \nacetylating specific target proteins. Despite their importance in bacterial physiology, the mechanisms 31 \nunderlying GNATs’ enzymatic and regulatory functions remain poorly understood. In this study, we 32 \nelucidated the structures of Escherichia coli PatZ, a type I GNAT , and investigated its ligand 33 \ninteractions, catalytic processes, and allosterism. PatZ functions as a homotetramer, with each 34 \nsubunit comprising a catalytic domain and a regulatory domain. Our findings reveal that the regulatory 35 \ndomain is essential for acetyltransferase activity, as it not only induces cooperative conformational 36 \nchanges in the catalytic domain but also directly contributes to the formation of substrate binding 37 \npockets. Furthermore, a protein structure-based analysis on the evolution of bacterial GNAT types 38 \nreveals a distinct pattern of the regulatory domain across phyla, underscoring the regulatory domain’s 39 \ncritical role in responding to cellular energy status.  40 \n 41 \nSIGNIFICANCE STATEMENT 42 \n 43 \nPost-translational modifications, particularly acetylation mediated by GCN5 -related N -44 \nAcetyltransferases (GNATs), play a crucial role in bacterial physiology. Protein acetyltransferase Z 45 \n(PatZ) is a key GNAT with diverse substrates, essential for understanding the bacterial acetylome. 46 \nThis study employs cryogenic electron microscopy, X-ray crystallography, and biochemical analyses 47 \nto elucidate the mechanistic regulation of Escherichia coli PatZ. Our high-resolution structures reveal 48 \nPatZ's homo-tetrameric architecture, with each subunit comprising regulatory and GNAT domains. 49 \nWe characterize ligand-PatZ interactions, demonstrating ligand-induced conformational changes that 50 \nfacilitate allosteric regulation of the catalytic domain. Furthermore, our analyses elucidate the 51 \nregulatory domain's contribution to substrate binding pocket formation, potentially enhancing 52 \nsubstrate specificity. Structure -based phylogenetic analysis provides insights into the evolution of 53 \ndiverse regulatory domains in the GNAT superfamily across bacterial taxonomy. This first visualization 54 \nof PatZ advances our mechanistic understanding of bacterial physiology, offering novel insights into 55 \nGNAT-mediated bacterial adaptations. 56 \n 57 \nHIGHLIGHTS 58 \n 59 \n- E. coli PatZ forms a homotetramer, with each subunit possessing a GNAT catalytic domain 60 \nand a regulatory domain. 61 \n- Cooperative binding of acetyl-CoA to the regulatory domains is a prerequisite for inducing the 62 \nstructural compatibility of the catalytic domain with a substrate. 63 \n- Diverse regulatory domains in GNATs evolved to adapt to varied metabolic conditions across 64 \nbacterial taxonomy.  65 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nINTRODUCTION 66 \n 67 \nPost-translational modifications (PTMs) play a crucial role in modifying protein structure, stability, 68 \nactivity, and their placement within the cell1–3. Acetylation stands out as one of the predominant PTMs, 69 \ninvolving the attachment of an acetyl group to the lysine residues or the protein's N -terminal4. This 70 \nmodification is a widespread phenomenon across all forms of life, facilitated either enzymatically by 71 \nacetyltransferases or non-enzymatically via high-energy compounds such as acetyl phosphate (AcP) 72 \nor acetyl-CoA (AcCoA)5–8. In eukaryotes, acetylation affects both histones and non-histone proteins9,10, 73 \nthereby influencing a broad array of cellular and physiological processes including transcription, phase 74 \nseparation, autophagy, mitosis, cellular differentiation, and neuronal activities 11–16 and disruption in 75 \nacetylation regulation is linked to cancer development and aging17,18. In prokaryotes, acetylation plays 76 \na significant role in regulating metabolism, modifying metabolic fluxes, cell growth, and survival19–21. It 77 \nalso plays a part in the regulation of gene expression by altering the activity of transcription factors 78 \naccording to environmental shifts 22. While the physiological significance of Nε-lysine acetylation is 79 \nwidely recognized in eukaryotes, its biological relevance in bacteria is still emerging. 80 \n 81 \nEnzymatic acetylation involves the addition of an acetyl group to lysine residues by enzymes known as 82 \nlysine acetyltransferases (KATs) 23. Based on sequence homology and biochemical characteristics, 83 \nKATs are classified into several families, including the p300/cAMP response element binding protein 84 \nbinding protein (CBP) family, Moz, Ybf2/Sas3, Sas2, Tip60 (MYST) family, and GCN5 -related N -85 \nacetyltransferase (GNAT) family 11,24,25. The MYST and p300/CBP families are found exclusively in 86 \neukaryotic cells, whereas the GNAT family includes orthologous proteins present in bacteria, 87 \neukaryotes, and archaea23. Approximately 65% of the GNAT domain superfamily is found in bacteria26. 88 \nRecent studies reveal that acetylation significantly impacts bacterial physiology, influencing translation 89 \nin Escherichia coli27, metabolic flux in Salmonella enterica28, and virulence in Francisella novicida29. 90 \nThese findings underscore the broad and vital impact of acetylation on bacterial physiology, ranging 91 \nfrom basic cellular processes to complex pathogenic mechanisms. 92 \n 93 \nThe catalytic GNAT domain is highly conserved across species. However, bacterial GNATs exhibit 94 \nunique regulatory domains which are diverse in different bacterial species8. These GNATs are classified 95 \ninto five types based on their architecture and arrangement of catalytic GNAT domain and regulatory 96 \ndomains (Figure 1a). Type I and II feature a homologous structure of NDP-forming acyl-CoA synthetase 97 \nin the regulatory domain, while Type III has a cAMP-binding, ACT (aspartate kinase, chorismate mutase, 98 \nTyrA) or NADP +-binding protein8. Types IV and V do not possess regulatory domain. Truncation or 99 \nmutations in these regulatory domains significantly diminished GNAT -mediated catalytic activity 8,30, 100 \nimplying that the regulatory and GNAT domains are functionally interdependent. In particular, protein 101 \nacetyltransferase Z (PatZ), also known as YfiQ, is one of the most well characterized Type I GNATs, 102 \nwith approximately 80% of its amino acids comprising the regulatory domain, and 20 % GNAT domain. 103 \nPatZ alters its oligomeric states and activity based on the presence of AcCoA, showing different patterns 104 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nin E. coli and S. enterica31,32. Recent studies about PatZ demonstrate that regulatory domain increases 105 \nthe acetyltransferase activity and that ligand binding to regulatory domain elevates AcCoA affinity to 106 \nGNAT domain31. However, the precise mechanisms remain unclear.  107 \n 108 \nIn this study, we elucidated the homotetrameric structure of functional PatZ using cryo -electron 109 \nmicroscopy (cryoEM). Each subunit of PatZ comprises four structural domains, binding to two AcCoA 110 \nmolecules, one ATP, and one phosphate. The unliganded -PatZ adopts an inactive conformation 111 \ncharacterized by a closed substrate binding pocket. Our findings reveal that the regulatory domains 112 \nundergo conformational changes upon ligand binding, altering their interactions with the GNAT domain. 113 \nSpecifically, the binding of AcCoA to the regulatory domain triggers a cooperative structural 114 \nrearrangement in the GNAT domain, which facilitates the binding of the AcCoA donor and organizes 115 \nthe substrate in close proximity for catalysis. Furthermore, our analysis suggests that PatZ coevolved 116 \nwith its substrate, with the regulatory domain playing a crucial role in substrate interaction. Additionally, 117 \nwe conducted a phylogenetic analysis of bacterial GNAT -mediated acetyltransferase families, which 118 \nprovides insights into the regulatory domain's role in activating distinct acetyl-PTM responses to various 119 \nmetabolic signatures. 120 \n 121 \n 122 \nRESULTS 123 \n 124 \nOverall architecture of E. coli PatZ 125 \nTo examine the biochemical and structural characteristics of PatZ, we purified the E. coli PatZ protein 126 \nand analyzed its acetyltransferase activity towards purified E. coli AcCoA synthetase (Acs) using 127 \nwestern blotting with an acetyl-lysine-specific antibody (Figure 1b). When supplied with AcCoA, PatZ 128 \nefficiently catalyzed the acetylation of Acs, consistent with findings from previous studies33,34. This PatZ-129 \nmediated enzymatic reaction is also significantly faster than the AcP-mediated non-enzymatic reaction 130 \n(Figure 1b)35, confirming that the prepared PatZ is active and functional. 131 \n 132 \nWe then delved into the three -dimensional structure of the unliganded form of PatZ to delineate its 133 \nmolecular architecture and assembly. The protein, with a protomer mass of approximately 100 KDa, 134 \npredominantly forms a homotetramer in solution, as inferred by size exclusion chromatography (Figure 135 \n1c). CryoEM imaging of the eluted PatZ protein revealed a tetrameric structure characterized by a 136 \ndiamond-shaped, four -fold symmetric architecture. We then achieved a resolution of 2.52 Å for a 137 \nconsensus map showing the composition of four distinct subunits. These units exhibit variable 138 \nresolution across the structure's periphery and thus structural features were enhanced by multiple 139 \nrounds of 3D -classification and variability analysis for each domain (Extended Data Figure 1a -e, 140 \nSupplementary Table 1). 141 \n 142 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nMolecular model was built by chasing the side densities and aligns closely with the cryoEM map, as 143 \ndemonstrated by high -quality statistical validation (Q -score=0.67, Extended Data Figure 1f). Four 144 \nsubunits are positioned in a cross-like arrangement, with a 180-degree rotation relative to adjacent units 145 \n(Figure 1d, Supplementary Video 1), giving the tetramer lengths of approximately 190 Å and 130 Å 146 \nalong the long and short axes, respectively. Notably each subunit is composed of four distinct structural 147 \ndomains. Reflecting on similarities with NDP -forming AcCoA synthetases (ACDs) 36 and proteins 148 \ncontaining GNAT motifs37, we designated these regions accordingly the CoA binding domain (CBD), 149 \nligase-CoA domain (LCD), ATP grasp domain (AGD), and GNAT domain (GNATD), sequentially 150 \narranged from the N- to the C-terminus (Figure 1e, Supplementary Video 1). 151 \n 152 \nPatZ presents a propensity to form a stable homotetramer structure, showcasing a sophisticated 153 \ninteraction among four protomers. The interactions exhibited significant contact areas with considerable 154 \nstabilization energies, notably 3103.4 Å2 with a ΔiG of -28.4 kcal/mol for the lateral interface and 619.5 155 \nÅ2 with a ΔiG of -12.0 kcal/mol for the axial interface. The formation of the lateral interface is achieved 156 \nthrough an antiparallel hetero -domain interaction between the CBD and LCD via dominant polar 157 \ninteractions (Figure 1f). Conversely, the axial interface with the CBDs stacking in a back-to-back fashion 158 \nat a 60-degree angle (Figure 1g). This mode of interaction enhances the stability through symmetric 159 \npredominant hydrophobic contacts. An attempt to disrupt tetramer form by mutating residues at the 160 \naxial interface led to significant protein aggregation. Collectively, our investigation into the PatZ protein 161 \ndelineates its acetyltransferase function within a uniquely stable tetrameric architecture. 162 \n 163 \nStructural basis of ligand binding to PatZ 164 \nBased on the structural homology with ACD1 36 and GNAT 37 proteins, we have identified four 165 \nhypothetical ligand binding pockets for two AcCoAs, ATP and phosphate (or AcP). To characterize the 166 \nrelationship between ligand and PatZ, we incubated purified PatZ with ATP, AcCoA, and phosphate, 167 \nand then analyzed it using cryoEM . We obtained a significantly higher resolution map converging at 168 \n1.99 Å (Q score=0.77, Extended Data Figure 2a -f, Supplementary Table 1). The overall shape and 169 \nsubunit arrangement of liganded PatZ remain identical but notably, we identified two AcCoA in each 170 \nprotomer, which is one in the N -terminal regulatory domain and the other in GNATD (Figure 2a,b, 171 \nSupplementary Video 2). We also identified phosphate densities located at each four lateral interfaces 172 \nbetween the LCD and CBD derived from different protomers (Figure 2a,b). Of note, we could not 173 \nvisualize ATP in AGD of the cryoEM map, which is potentially reasoned by structural dynamics in the 174 \ndomain. Therefore, we performed complementary X-ray crystallographic experiments for the AGD and 175 \nconfirmed bound ATP in AGD resolution with 2.24 Å (Figure 2a,b, Extended Data Figure 2g, 176 \nSupplementary Table 2). Collectively, we successfully visualized all possible ligand binding structures.  177 \n 178 \nAcCoA in catalytic GNATD: Within the PatZ subunits, each GNATD adopts a zigzag configuration, 179 \noriented outward to maximize its accessibility and functional flexibility. The structural composition of the 180 \nGNATD includes five α-helices and eight β-strands, forming a donut-shaped tunnel that facilitates the 181 \nconcurrent orientation of AcCoA and the substrate (Supplementary Video 3). This arrangement is 182 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\npivotal for the enzymatic activity, directing AcCoA and substrate into a productive alignment. The 183 \nGNATD features a positively charged surface that binds to AcCoA, and a negatively charged surface 184 \nthat assists in positioning acceptor lysine residues. AcCoA is strategically placed within a V -shaped 185 \ncleft, bordered by β -strands, ensuring precise orientation. The pyrophosphate group of AcCoA is 186 \nanchored through interactions with the phosphate -coordinating loop (P-loop38, G822-G824) including 187 \nwater-mediated hydrogen bonding and a phosphate group on the ribose, which is secured by K860 188 \n(Figure 2c). Moreover, the acetate group of AcCoA, crucial for triggering enzymatic reactions, is situated 189 \nnear E809. This residue is identified as a potential catalytic site32 and is in close proximity to the putative 190 \nacceptor lysine binding site, underscoring its significance in the enzymatic process. 191 \n 192 \nAcCoA in N-terminal CBD: The PatZ enzyme distinguishes itself through the inclusion of a regulatory 193 \ndomain alongside its catalytic domain, a feature not fully elucidated in terms of its mechanistic role. We 194 \nvisualized a well-defined AcCoA density within the N-terminal CBD (1-285) of PatZ. Notably, the CoA 195 \nportion of AcCoA is positioned to project outward, while the acetyl group is nestled within the inter -196 \nsubunit cleft. The binding pocket is characterized by its positive charge and an elongated, narrow 197 \nmorphology, tailored to interact with AcCoA's three phosphate groups. This interaction is further 198 \nreinforced by the pocket's design to accommodate the distinct components of AcCoA through a network 199 \nof hydrogen bonds including water -mediated interactions (Figure 2d). Particularly, the positively 200 \ncharged residues (K23, R26 and R81) within the pocket engage in electrostatic interactions with 201 \nAcCoA's phosphate groups, underscoring the specificity and efficiency of the binding process. 202 \n 203 \nPhosphate located in intersubunit interface: We have identified a significant density in the interface 204 \nbetween each CBD and LCD derived from different protomers. Considering the homology with ACD136, 205 \nthis density can be attributed to phosphate. The phosphate binding site is located at the tip of the α -206 \nhelices originating from the LCD and CBD, and is further stabilized by additional loops. The phosphate 207 \nis spatially adjacent to AcCoA, which resides within the CBD (Figure 2d). Since the phosphate ion is 208 \ndeeply embedded inside the structure, stabilizing a lateral dimer. Of note, this phosphate can be 209 \nconverted to AcP during ATP synthesis pathway for ACD enzyme 39. Through structural analysis, we 210 \nfound that although PatZ's regulatory domain is incapable of enzyme catalysis, it still binds phosphate 211 \nin the same location as ACD1. 212 \n 213 \nATP in ATP grasp domain: The ATP grasp domain (AGD, 451 -709) encompasses the ATP binding 214 \npocket. Since the dynamic nature of the AGD complicates the visualization of ATP density in cryoEM. 215 \nConsequently, X-ray crystallography was employed, successfully confirming the presence of ATP within 216 \nthe AGD's binding pocket at a 2.24 Å resolution. This structure shows that the ATP binding site is 217 \nnestled in a cleft located in the central AGD, engaging in various interactions with surrounding amino 218 \nacids (Figure 2e). Despite the presence of 1 mM magnesium during the crystallization process, no 219 \nelectron density map was observed. Instead of a divalent ion, the triphosphate moiety of ATP is 220 \nstabilized by two histidines (H531, H676) in AGD. 221 \n 222 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nAcCoA binding activates GNATD 223 \nTo understand how AcCoA binding activates catalytic GNATD and the chemical basis of its catalytic 224 \nmechanism, we compared GNATD structures between the apo- and AcCoA-bound states. Binding of 225 \nAcCoA to GNATD induces significant spatial changes, particularly in the substrate gating motif (V746-226 \nT763, hereafter, gating motif), which are located near the substrate access channel and undergo a 227 \nnotable secondary structural transition (Figure 3a, Supplementary video 3). In apo-PatZ, the β-sheet of 228 \nthe gating motif is extended, covering the substrate binding sites of GNATD. However, in the presence 229 \nof AcCoA, this region transforms into a loop and an α-helix, establishing direct interaction with AcCoA 230 \n(Figure 3b) and opening a substrate binding pocket (Figure 3c). This structural observation indicates 231 \nthe extended β-sheet of gating motif sterically obstructs the interaction between donor AcCoA and the 232 \nacceptor substrate. AcCoA binding triggers the rearrangement of the gating motif, removing the physical 233 \nbarrier between AcCoA and the substrate. These structural rearrangements can be crucial to organize 234 \nthe AcCoA donor and the substrate in catalytic proximity. Therefore, we conclude that AcCoA binding 235 \nis required for GNATD by switching the gating motif into an active structural conformation. 236 \n 237 \nBased on the active conformation of GNATD, we investigated the critical residues that potentially 238 \ncontribute to enzyme activity and substrate binding. By focusing on the distinctive donut hole shape of 239 \nthe GNATD, we observed that the acetyl group of the acetyl-donor AcCoA is positioned at the center of 240 \nthis hole (Figure 3d), oriented towards the substrate binding site. This region contains several charged 241 \nresidues, and we created alanine mutants for four key amino acids (R754, F756, E809, I846) and 242 \ncompared their enzyme activity to that of the wild -type. The results showed that mutations in any of 243 \nthese four residues led to a significant reduction in enzyme activity (Figure 3e). Based on these 244 \nfunctional tests and structural analysis, we confirmed that the acetyl group of AcCoA and the acetyl -245 \nacceptor lysine are strategically positioned around the donut hole of the GNATD, playing a crucial role 246 \nin the acetyl-transfer reaction. 247 \n 248 \nTo gain a deeper chemical understanding of the enzyme mechanism, we compared our structure with 249 \nthat of the GNATD of Streptomyces lividans PatA (slPatA), which has been structurally characterized 250 \nin complex with S. enterica Acs (seAcs) (PDB ID: 4U5Y)40 . The GNATD of slPatA is structurally similar 251 \nto that of PatZ GNATD, with an RMSD of 1.12 Å (Extended Data Figure 3a). By integrating structural 252 \ninformation, we identified the arrangement of key catalytic residues in PatZ, including E809 and the 253 \ncarbonyl oxygens of F810 and I846 (Figure 3f). Notably, these residues are highly conserved within the 254 \nGNAT family. E809, in particular, is recognized as a crucial catalytic residue (Extended Data Figure 3b). 255 \nOf note, this glutamate residue can activate a water molecule, which coordinated by E123PatA, carbonyl 256 \noxygen of V124PatA, amide nitrogen of E160 and K609 Acs, to remove a proton from the lysine amine 257 \ngroup, facilitating a nucleophilic attack on the carbonyl carbon of enzyme -bound AcCoA (Figure 3f). 258 \nTaken together, the structural similarities between slPatA and E. coli PatZ, coupled with the conserved 259 \nnature of the catalytic residues, underscore the mechanistic insights into the enzymatic function of PatZ 260 \n(Figure 3g). These findings imply the critical roles of E809 and the amino acid backbones in facilitating 261 \nthe acetyl-transfer reaction, advancing our understanding of the GNAT family’s catalytic mechanisms. 262 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 263 \nAllosteric regulation for PatZ 264 \nNext, to investigate the functional and mechanistic association between the regulatory domain and 265 \ncatalytic GNATD, we generated domain-wise truncated PatZ mutants and examined their activity. The 266 \nAGD-truncated mutant (trAGD) produced soluble aggregates, while the other truncated mutants 267 \n(trCBD-LCD, GNATD-only and regulatory domain-only (RD-only)) were properly folded as evidenced 268 \nby size exclusion chromatography (Figure 4a). As expected from our structural data, the RD -only 269 \nmutant formed a tetramer, whereas the trCBD -LCD and GNATD-only mutants existed as monomers 270 \nbased on their elution profiles. As anticipated, the RD -only mutant without catalytic module abolished 271 \nenzymatic activity. Interestingly, both the GNATD-only mutant and the domain-wise truncation mutants 272 \nof the regulatory domains also lost their acetyltransferase activity towards Acs (Figure 4b). It is 273 \nnoteworthy that the trAGD mutant, which showed aggregation, likely experienced a loss of activity 274 \nrelated to the instability of its truncated structure (Extended Data Figure 4a). The loss of enzymatic 275 \nactivity in both the GNATD -only and truncated regulatory domain mutants suggests the necessity of 276 \noligomeric conformation and highlights the critical interplay between these domains for the 277 \nacetyltransferase function of PatZ.  278 \n 279 \nTo investigate whether and how AcCoA binding to the regulatory domain affects PatZ activity, we 280 \nanalyzed the AcCoA binding affinity of PatZ using isothermal titration calorimetry (ITC). As anticipated 281 \nfrom a previous study on S. enterica Pat31 and our structural data in this study (Figures 2 and 3), AcCoA 282 \nbinding to wild-type PatZ exhibited a biphasic curve, best fitting to a two-site binding model. One binding 283 \nsite demonstrated a much stronger affinity (K d1 = 0.60 μM) compared to the other (K d2 = 3.98 μM) 284 \n(Figure 4c). However, when residues R26 and R81 within the AcCoA-binding pocket of PatZ (see Figure 285 \n2d) was mutated to Glu, disabling AcCoA binding to the RD, the resulting RR mutant of PatZ showed 286 \none-site binding (Kd = 12.10 μM), similar to, but with lower affinity than, the RD -only form (Kd = 5.15 287 \nμM). These kinetic data suggest that the RD has a higher AcCoA binding affinity than the GNATD and 288 \nthat binding of AcCoA to one site increases the affinity of the other site, indicating positive cooperativity. 289 \nNotably, the monomeric GNATD -only form exhibited a binding affinity (K d = 8.13 μM) similar to the 290 \ntetrameric RR mutant, indicating that the AcCoA binding affinity of GNATD is not significantly affected 291 \nby oligomerization. 292 \n 293 \nNext, we conducted the PatZ activity assay by measuring the free sulfhydryl group of CoA released 294 \nafter the acetyltransferase reaction. The formation of 2-nitro-5-thiobenzoate (TNB2-) from the reaction 295 \nbetween the free sulfhydryl group and DTNB (5,5'-dithio-bis-(2-nitrobenzoic acid)) was recorded at 412 296 \nnm. Consistent with previous findings for ecPatZ32 (Hill coefficient = 7.91) and sePatZ31 (Hill coefficient 297 \n= 2.2), wild -type PatZ exhibited a sigmoidal activity curve, indicative of positive cooperativity (Hill 298 \ncoefficient = 2.21; Khalf = 4.20 μM) (Figure 4d). However, the RR mutant exhibited a typical Michaelis-299 \nMenten curve and had a significantly lower AcCoA affinity (Km = 11.78 μM) compared to wild-type PatZ. 300 \nThese results confirm that AcCoA binding to the regulatory domain not only increases GNATD's affinity 301 \nfor AcCoA but is also essential for the proper allosteric regulation of PatZ's enzymatic activity.  302 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 303 \nTo understand the mechanistic relationship with AcCoA-binding between the regulatory domains and 304 \nGNATD, we analyzed the domain-wise structural dynamics of PatZ upon ligand binding using single 305 \nprotomer heterogeneity from our cryoEM images. PatZ displayed varying degrees of structural 306 \ndynamics with and without AcCoA (Figure 4e, Supplementary Video 4). Specifically, apo-PatZ exhibited 307 \nsignificantly higher structural heterogeneity compared to the ligand-bound form, as also evidenced by 308 \nresolution improvement from 2.56 Å to 1.99 Å. The AGD's motion was less correlated and continuous 309 \nregardless of AcCoA binding. However, we identified three distinct classes (class I-III, Extended Data 310 \nFigure 4b) of domain interfaces of the LCD with ~15 Å RMSD in apo -PatZ, while the AcCoA -bound 311 \nstructure showed a well-converged single conformation (Figure 4f). These structural changes in apo -312 \nPatZ resulted in a relaxed axial intersubunit space between the LCD of one subunit and the CBD of the 313 \nneighboring subunit. To characterize changes in the interface, we measured the distance between S156 314 \nand R343 of the neighboring protomer, forming part of the ligand binding pocket (Figure 4g, Extended 315 \nData Figure 4c). In the apo -state, this distance expanded to 16.9 Å. In the ligand bound form, the 316 \ndistance converged to 11.9 Å. Notably, the class III map in apo -PatZ displayed attributable density to 317 \nphosphate with an RMSD of 1.2 Å compared to the AcCoA-bound conformation. Therefore, our analysis 318 \nindicates that AcCoA binding in the CBD contributes to stabilizing the complex and enforcing a stably 319 \nclosed intersubunit conformation. 320 \n 321 \nInterestingly, the GNATD itself displays well-defined static structures in both the apo- and ligand-bound 322 \nstates. However, binding of AcCoA to PatZ induces significant domain -wise movement in GNATD, 323 \nspecifically a ~37° rotation and a ~40° tilt relative to the regulatory domain from the apo - to AcCoA-324 \nbound conformation (Figure 4h, Supplementary Video 4). We identified that the regulatory domains 325 \nalter their specific interactions with the GNATD upon ligand-induced conformational transition. In apo-326 \nPatZ, the N-terminal end of CBD (Figure 4i) interacts with the β-sheet of gating motif, anchoring GNATD 327 \nin a down-position. Conversely, in the ligand-bound conformation, the AGD and the interdomain loop 328 \n(E451-S561) establish new specific interactions with the hinge region (E736, Q772, N769) of GNATD, 329 \nstabilizing GNATD in an up-position (Figure 4h). 330 \n 331 \nImportantly, in the apo -PatZ, the tethered configuration limits the space between GNATD and CBD, 332 \nresulting in the closure of the substrate access channel, with the β -sheet of gating motif physically 333 \nobstructing the interaction between donor AcCoA and the substrate (Figure 4j, left panel). Binding of 334 \nAcCoA to both the CBD and GNATD opens the substrate access channel and triggers the 335 \nrearrangement of the gating motif into an active conformation (Figure 4j, right panel). Taken together, 336 \nthe regulatory domain is essential for the activation of GNATD, and AcCoA binding to both the CBD 337 \nand GNATD positively cooperates in allosteric regulation to facilitate PatZ's enzymatic reaction, which 338 \nis in accordance with the ITC and DTNB results (Figure 4c,d). Notably, considering that the close 339 \nconformation of the regulatory domain (class III in apo-PatZ) still displays a down-position of GNATD, 340 \nit is logical to conclude that AcCoA binding to GNATD and the switching interaction from the regulatory 341 \ndomain are both prerequisite events for PatZ activation. 342 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 343 \nRegulatory domain directly shapes Acs binding pocket, enhancing substrate interaction. 344 \nUpon elucidating a cooperative mechanism between the GNATD and regulatory domains of PatZ for 345 \nligand-induced substrate gating, we extended our investigation to explore whether the regulatory 346 \ndomain directly influences binding or specificity of a protein substrate. AlphaFold41 predicted the reliable 347 \nmodels showing monomeric interaction within the PatZ-Acs complex structure (Extended Data Figure 348 \n5). These predictions illustrated that Acs interacts with PatZ and notably, the Acs K609 residue, a known 349 \nacetylation site, is oriented towards GNATD (Figure 5a) and is positioned within the putative acceptor 350 \nlysine binding pocket, which is consistent with our experimental analysis (Figure 5a,b). This intricate 351 \ninteraction of Acs is established by an orchestration with the CBD, LCD and GNATD of PatZ. 352 \n 353 \nSignificantly, the predicted PatZ -Acs structure underscores that the regulatory domain forms a 354 \ndistinctive groove and binding surface, accommodating the C -terminal domain of Acs and fostering 355 \nhighly complementary polar interactions (Figure 5b). These specific residues exhibit robust coupled 356 \nvariance, indicative of residue-level coevolution between Acs and the regulatory domain of PatZ. This 357 \ncoevolution suggests a finely tuned evolutionary adaptation, underscoring the additional importance of 358 \nthe regulatory domain for PatZ's functional integrity. 359 \n 360 \nTo validate the impact of regulatory domain and Acs interaction on enzyme activity, we identified and 361 \nmutated coevolved residues at the interface between Acs-CTD and the regulatory domain of PatZ. We 362 \nengineered R395A/Y398A and N227A/K228A mutants of PatZ (Figure 5c) and evaluated their enzyme 363 \nactivity compared to the wild-type over time. Our results disclosed a pronounced decrease in enzyme 364 \nactivity for both mutants (Figure 5d), signifying the critical role these residues play in maintaining 365 \nenzymatic function. This significant reduction in activity underscores the importance of the regulatory 366 \ndomain in facilitating proper substrate binding and, consequently, effective catalysis. 367 \n 368 \n 369 \nDISCUSSION 370 \n 371 \nWe characterized multiple ligand -PatZ interactions and demonstrated that ligand -induced 372 \nconformational changes facilitate the allosteric regulation of the catalytic activity (Figure 4, 6a). Our 373 \ninvestigation delineates PatZ’s working mechanism and highlights key aspects of how ligand binding 374 \ninfluences PatZ activity and regulation (Figure 6a). 375 \n 376 \nGating motif contributes to auto-regulation of GNATs 377 \nLocated near the substrate binding site in GNATD, the gating motif (V746-T763) undergoes significant 378 \nstructural changes, demonstrating the mechanism of opening and closing a passage that connects the 379 \ndonor AcCoA and acceptor protein substrate (Figure 4h-j, 5). In the absence of AcCoA, the gating motif 380 \nsterically occludes substrate binding sites and masks the passage, indicating inhibited conformation 381 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n(Figure 4h-j). Upon AcCoA binding, the gating motif coordinates with the AcCoA to expose the substrate 382 \nbinding sites and aligns the proximity between AcCoA and protein substrates, indicating activated 383 \nconformation (Figure 4h -j). AcCoA induced -structural changes in GNATs have been previously 384 \ndiscussed in the studies of RimL from S. typhimurium42, Rv0819 from M. tuberculosis43 and ssPat from 385 \nSulfolobus solfataricus44. RimL and ssPat are GNATD only enzymes without regulatory domain (Type 386 \nIV GNAT), while Rv0819 is a tandem-repeated GNAT enzyme (Type V GNATs). In these studies, the 387 \nhomologous region with a gating motif is referred to as α1 -α2 loop, mobile loop or bent helix, which 388 \nremain unresolved in the absence of AcCoA due to their intrinsic dynamic features. This loop becomes 389 \nrigidly ordered by AcCoA binding, resulting in the formation of a substrate binding pocket, similar to the 390 \ngating motif of PatZ. Notably, the mutation study of ssPat has shown that the bent helix is essential for 391 \nenzyme activity.  392 \n 393 \nImportantly, this structural component, the gating motif in GNATs, is not only present in prokaryotes but 394 \nalso preserved in eukaryotes, including humans (Extended Data Figure 7), which suggests a common 395 \nmechanism of GNATs’ auto-regulation across species. Collectively, based on the clear visualization of 396 \nthe inhibitory and active conformations of GNATD in PatZ, we propose an auto-regulatory mechanism 397 \nof GNATs mediated by the gating motif. Interestingly, despite the high structural similarity of GNATs, 398 \nthe gating motif exhibits relatively low sequence similarity45 (Extended Data Figure 7). Since the gating 399 \nmotif also contributes to the formation of the substrate binding pocket, it is tempting to speculate that 400 \nthe gating motif may be involved in substrate specificity, which requires further studies. 401 \n 402 \nRegulatory domain interaction mechanistically regulates PatZ activity 403 \nWe characterized that ligand binding to the regulatory domain significantly stabilizes intersubunit 404 \ninterface and rigidifies PatZ's homotetrameric architecture (Figure 4g). Concurrently, this 405 \nconformational stabilization of the regulatory domain is correlated with the activation process in the 406 \ngating motif induced by AcCoA binding to GNATD (Figure 4i). These cooperative structural events result 407 \nin the domain-wise conformations of the up- and down-positioning of the GNATD. The gating motif of 408 \nGNATD interacts with the CBD in the absence of AcCoA, leading to the down -positioning of GNATD 409 \n(Figure 4i, left panel). In this down position, the gating motif tethered by the regulatory domain illustrates 410 \na restricted space for the substrate access channel (Figure 4j, left panel), indicating an inhibitory 411 \nconformation. Upon AcCoA binding, the gating motif loses its interaction with CBD, allowing GNATD to 412 \nrefresh its interaction with AGD and adopt an up-positioned conformation (Figure 4i, right panel). This 413 \nposition creates a widely exposed space between GNATD and CBD, allowing protein substrates access 414 \nto the substrate binding site (Figure 4j, right panel), indicating an activated conformation. 415 \n 416 \nInterestingly, the stabilized structure with phosphate bound PatZ (Extended Data Figure 4b,c) displays 417 \nthe down position of GNATD and the inactive conformation of the gating motif. The regulatory domain 418 \nof this structure is identical to that of the AcCoA -bound structure, implying that the stability of the 419 \nregulatory domain alone cannot transform GNATD into its active conformation. GNATD alone does not 420 \nalso have enzymatic activity and thus suggest that AcCoA binding to both CBD and GNATD is a 421 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nprerequisite for activating PatZ. Importantly, we (Figure 4c,d) and others 31 have demonstrated the 422 \ncooperative relationship of AcCoA binding between GNATD and CBD. AcCoA binding to the wild-type 423 \nGNATD is approximately 2 times higher than to the GNATD -only protein and 6.6 times higher to the 424 \nregulatory domain than to GNATD (Figure 4c). These findings collectively suggest that AcCoA binding 425 \nto CBD reduces the dynamics of the regulatory domain, enhances the AcCoA binding to GNATD, 426 \nfacilitates the rearrangement of the gating motif, and activates the enzyme. 427 \n 428 \nTherefore, our study elucidates the active and inactive conformations in the cooperation between 429 \nGNATD and the regulatory domain and highlights PatZ’s activity regulation mechanism. This elegant 430 \nmechanism employs a dual strategy that switches between active and inactive conformations and 431 \nallosterically modulates enzyme activity upon cooperative AcCoA binding. 432 \n 433 \nRegulatory domain contributes PatZ’s substrate specificity 434 \nPrior studies have highlighted the unique specificity of PatZ for Acs32,46, and it has been also observed 435 \nthat strains lacking PatZ do not produce acetylated Acs 47. Through our observations, we identified a 436 \nstructural opening event in GNATD, in cooperation with CBD, that forms an Acs-specific binding pocket 437 \n(Figure 5). These findings validate additional functional layers of the regulatory domain. Moreover, it is 438 \nevident that GNATD alone in PatZ lacks enzymatic activity toward Acs 40 (Figure 4b), and point 439 \nmutations in the regulatory domain that disrupt Acs interaction significantly reduce Acs acetylation 440 \n(Figure 5c,d). Our results further demonstrate that Acs binds complementary to the substrate binding 441 \nsite formed by both GNATD and the regulatory domain (Figure 5a,b). This is supported by the 442 \nobservation that E. coli-derived YiaC, which lacks a regulatory domain (GNAT type IV), is inactive to 443 \nAcs (Supplementary Data Figure 8). The residues involved in Acs-PatZ interaction are highly conserved, 444 \nsuggesting a finely tuned evolutionary adaptation that underscores the regulatory domain's critical 445 \nimportance for PatZ's function concerning Acs. 446 \n 447 \nWhile we elucidate substrate-specific mechanisms, it is noteworthy that PatZ is recognized for its ability 448 \nto acetylate a diverse array of protein substrates within the bacterial proteome, with an estimated around 449 \n1000 substrates compared to around 20~600 for GNATD -only enzymes like YjaB, YiaC, RimI and 450 \nPhnO48. Although these acetyl -substrate repertoires overlap significantly, PatZ exhibits a unique 451 \ncapability for the specific targeting of substrates32,46. 452 \n 453 \nOur study implies that PatZ employs dual modes of substrate recognition: a general acetyltransferase 454 \nfunction for exposed lysines across a broad spectrum of proteins and a specific mechanism through 455 \ncooperative substrate recognition involving both GNATD and the regulatory domains (Figure 6a). This 456 \ndual functionality highlights the sophisticated regulatory mechanisms that enable PatZ to achieve both 457 \nsubstrate specificity and enzymatic versatility. 458 \n 459 \nDiverse roles of regulatory domains in GNATs family for bacterial physiology 460 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nProtein acetylation plays crucial roles in cellular physiology by impacting various processes and 461 \nregulatory mechanisms49. Present in all living organisms across the Bacteria, Archaea, and Eukarya 462 \ndomains50, protein acetyltransferases, including GNATs suggest that protein acetylation has existed 463 \nsince the earliest stages of life, predating the Last Universal Common Ancestor (LUCA) more than 4 464 \nbillion years ago51. Despite the vast evolutionary divergence from LUCA, essential cellular functions 465 \nsuch as genetic information processing and core metabolic pathways have been conserved across 466 \nphylogeny52. 467 \n 468 \nOur study reveals that the mechanistic coupling of AcCoA binding to the regulatory domain modulates 469 \nthe activity of the Type I GNATs, such as PatZ. Given that unique types of regulatory domains in other 470 \nGNAT’s can bind different ligands, this variation suggests that bacteria regulate GNATs’ activity in 471 \nresponse to diverse metabolic conditions (Figure 6b). We conducted a protein structure-based 472 \nphylogenetic analysis against the AlphaFold Protein Structure Database53  to explore the distribution of 473 \nGNAT types  in bacteria (Extended Data Figure 6). Notably, we found that while bacteria can 474 \nsimultaneously possess multiple types of GNATs, Type IV GNATs, which consist solely of the GNATD, 475 \nexhibit a ubiquitous distribution across the bacterial domain (Extended Data Figure 6e ,f). In contrast, 476 \nwe identified a distinct pattern where other types of GNATs with unique regulatory domains are 477 \npredominantly found within specific phyla (Extended Data Figures 6e,f). 478 \n 479 \nType IV GNATs, processing a single catalytic GNAT domain, are ubiquitously present in bacteria 480 \n(Extended Data Figure 6e,f), where they regulate nucleoid organization 54, transcription55, and central 481 \nmetabolic pathways56. This ubiquity implies GNATs’ involvement in regulating housekeeping processes. 482 \nIn contrast, Type I, II, and III GNATs possess additional regulatory domains (Figure 6b, Extended Data 483 \nFigure 6b-d), likely enabling them to sense various environmental conditions and tightly regulate genetic 484 \ninformation flow and metabolic fluxes in response to nutrient availability and cellular energy status. 485 \n 486 \nFor instance, in E. coli, the Type I GNAT PatZ uses AcCoA not only as an acetyl -donating substrate 487 \nbut also as a ligand binding to its regulatory domain (Figure 2), thus allosterically regulating its GNAT 488 \nactivity (Figure 4). One of the most studied Type II GNATs is PatA from S. lividans57. While the ligands 489 \nbinding to its regulatory domain are not well understood, predictions using AlphaFold41 and AlphaFill58 490 \nrevealed that AcCoA and ATP bind to PatA’s regulatory domain (Figure 6b). Previous studies have 491 \nreported that Type III GNATs have their activity regulated by the binding of amino acids, cAMP, and 492 \nNADP+ to their regulatory domains59–61. Although different types of GNATs adopted various ligands as 493 \nregulatory cues, these ligands generally represent nutritional and cellular energy status62. 494 \n 495 \nInterestingly, the activity of Acs is regulated by acetylation via GNATs in most, if not all, bacterial species 496 \nstudied so far4. AcCoA is a crucial metabolite in central metabolism, acting as a crossroad in pathways 497 \nsuch as glycolysis, gluconeogenesis, the tricarboxylic acid cycle, the glyoxylate bypass, lipid 498 \nmetabolism, and amino acid synthesis63. Nearly all life forms have evolved to adopt AcCoA as a key 499 \nmetabolic intermediate, making it essential to maintain its levels for supporting all cellular processes. 500 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nWhile different bacterial phyla have adopted different types of structures of regulatory domains (Figure 501 \n6b-d), a common function of GNATs appears to be the regulation of housekeeping processes by 502 \nsensing diverse cellular cues. 503 \n  504 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nMATERIAL AND METHODS 505 \n 506 \nConstructs design and molecular cloning 507 \nThe full-length or truncated PatZ gene (UniProt ID: P76594) was cloned into the pETDuet -1 vector 508 \n(Novagen) with a 6xHis tag at the C-terminus. The full-length Acs gene (UniProt ID: P27550) was cloned 509 \ninto the pETDuet-1 vector with an N -terminal 6xHis tag. All mutants were generated by site -directed 510 \nmutagenesis.  511 \n 512 \nProtein expression and purification 513 \nThe recombinant DNA is transformed into the patz, pta deletion ER2566 strain. The transformed cells 514 \nwere grown in LB medium at 37 °C until the O.D 600 value reached 0.6~0.8. The temperature was then 515 \nlowered to 17 °C, and 1 mM IPTG was added to induce protein expression. After 16 hours of induction, 516 \nthe cells were harvested by centrifugation and the medium was completely discarded for storage at -517 \n80 °C. The harvested cells were disrupted using Emulsiflex C3 (Avestin) and the cell debris was 518 \nremoved by centrifugation at 20,784 g for 30 min. The supernatant is filtered before purification by FPLC. 519 \nAnd the filtered samples were purified by Ni-NTA affinity chromatography using Histrap HP pre-packed 520 \ncolumns (Cytiva). After washing with wash buffer (25 mM Tris -HCl pH 7.4, 500 mM NaCl, 20 mM 521 \nimidazole, 1 mM MgCl 2, 10% glycerol) elution was performed with elution buffer (20 mM HEPES pH 522 \n7.4, 500 mM NaCl, 1 mM MgCl2, 10% glycerol, 1 mM DTT) with 300 mM Imidazole. The purified protein 523 \nsamples were then further purified by size exclusion chromatography (SEC) using a Superose 6 524 \nIncrease column (Cytiva) in a buffer containing PBS with additional 100 mM NaCl. Acs was cultured in 525 \nTB medium under the same conditions. The purification procedure followed the same protocol: After 526 \nextensive washing with wash buffer (25 mM Tris -HCl pH 7.4, 500 mM NaCl, 40 mM imidazole, 10% 527 \nglycerol), elution was carried out using elution buffer (25 mM Tris-HCl pH 7.4, 500 mM NaCl, 300 mM 528 \nimidazole, 10% glycerol). and then further purified by SEC using a HiLoad 16/600 superose 6 pg (Cytiva) 529 \nin a buffer containing 25 mM Tris-HCl pH 7.4, 500 mM NaCl, 5% glycerol. 530 \n 531 \nCryoEM specimen preparation and imaging condition 532 \nPurified protein sample was adjusted to 1.5 mg/mL and vitrification was conducted using the Vitrobot 533 \nsystem (ThermoFisher). 3 μL samples were applied to the discharged UltrAuFoil R1.2/1.3 gold 300 534 \nmesh (Quantifoil). Grids were blotted for 3 sec and plunged into liquid ethane at 100% humidity at 8 °C. 535 \nThe grids were roughly screened on a Glacios operated at 200 keV (cryoTEM, ThermoFisher) and 536 \nequipped with a Falcon 4 direct electron detector. The nicely vitrified grids were placed on a 300 keV 537 \nTitan Krios G4 (ThermoFisher) equipped with a Falcon 4i direct electron detector. Data was acquired 538 \nin EER format at a pixel size of 0.723 Å/pixel using aberration-free image shift (AFIS) to accelerate the 539 \ndata acquisition. and total exposure dose was 50 e/A2 with -0.8 to -1.5 μm defocus range. More details 540 \nabout imaging conditions are described in Supplementary Table 1. 541 \n 542 \nCryoEM image processing 543 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nThe EM data processing workflow for all datasets is illustrated in Extended Data Figures 1 and 2. The 544 \nprocessing was conducted using CryoSPARC v4.4.1 packages, and a comparable strategy was 545 \nemployed for all datasets. Firstly, the beam -induced motion of the movies was corrected using Patch 546 \nmotion correction, and the contrast transfer function (CTF) of each micrograph was calculated using 547 \nPatch CTF estimation. Some micrographs were excluded due to insufficient resolution of the CTF and 548 \ndefocus range. Subsequently, about 3,000 particles were selected via blob-based picking from a subset 549 \nof 30 micrographs, resulting in the generation of 2D templates. Finally, a considerable number of 550 \nparticles were automatically selected from the entirety of the micrographs using the templates. The 551 \nextraction of particles was conducted using a box size of 448 X 448 pixels. Subsequently, several 552 \niterations of 2D classification were performed, and 3D volumes were generated without alignment 553 \nthrough Ab -initio reconstruction. The optimal volume class with D2 symmetry was subjected to 554 \nHomogeneous 3D refinement. To enhance the overall quality of the particles and volumes, Topaz 555 \nparticle picking and Reference-based motion correction (beta) were applied based on the initially refined 556 \nmap. Subsequently, Final maps were generated, followed by global and local CTF correction to 557 \nenhance the map. 558 \n 559 \nFor asymmetric unit analysis, each particle was expanded according to D2 symmetry. The optimal 560 \nvolumes for each domain and subunit were classified through multiple rounds of 3D classification and 561 \nsubjected to local refinement using the corresponding masks without any symmetry enforcement. 562 \nThese refinements significantly improved domain resolution and features. Structural heterogeneity was 563 \ncharacterized by iterative 3D classification and 3D variability analysis. To generate representative final 564 \nmaps, locally refined maps were aligned into the consensus refined map and patched using the “vop 565 \nmaximum” command in UCSF ChimeraX 64 . These focused refinement maps and the merged maps 566 \nwere subsequently used for model building, refinement, and analysis. 567 \n 568 \nModel building and refinement 569 \nInitial models were generated using AlphaFold and subsequently fitted to the map using ISOLDE. 570 \nManual model fitting was then performed using the Coot program, followed by model refinement using 571 \nReal-space refinement in Phenix. The refinement results and model quality were numerically assessed 572 \nthrough Real-space refinement output values and MolProbity scores. The structure of liganded -PatZ 573 \nwas built using the structure obtained from apo-PatZ as a template, and model refinement was carried 574 \nout following the same methodology as for apo-PatZ. 575 \n 576 \nX-ray crystallography, data processing and refinement 577 \nAGD of PatZ (T463 -S695) was subcloned into pET28a and overexpression and purification were 578 \nconducted using the same method but with an SEC buffer containing 20 mM HEPES -NaOH pH 7.5, 579 \n150 mM NaCl, 1 mM MgCl2, 1 mM DTT. Concentration of purified samples was adjusted to 10 mg/mL 580 \nand co-crystallized with 5 mM ATP. Initial crystallization screening was conducted by using a Mosquito 581 \nrobot (TTP Labtech) and a single, appropriate size of crystals appeared at 0.2 M potassium sodium 582 \ntartrate, 20 % (w/v) PEG 3350. 20 % ethylene glycol was added as a cryo-protectant and flash frozen 583 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nin liquid nitrogen. Crystal diffraction was carried out at the Pohang accelerator laboratory 5C (PAL-5C) 584 \nbeamline (wavelength= 0.97942 Å)65 on 100K temperature. Diffraction data sets were processed and 585 \nscaled with the programs HKL2000 66 and imosfilm 67 . The phasing information was solved by a 586 \nmolecular replacement (MR) method using the cryoEM structure. MOLREP68, REFMAC569 , and COOT 587 \nwere used for MR, structure refinement and further modeling, respectively. Figures were prepared using 588 \nPYMOL and ChimeraX. The Ramachandran plot analysis of the final model revealed that 96.44% of 589 \nresidues were in favored regions, 3.34% in allowed regions, and 0.22% in disallowed regions. 590 \n 591 \nIn vitro acetylation assay: Western blot   592 \nAcs (1.3 μM) was incubated with wild-type or mutant PatZ (425 nM) in the presence of 1 mM AcCoA 593 \n(Cayman) or 1-10 mM of AcP (Merck) in an assay buffer (25 mM sodium phosphate pH 8.0, 0.5 mM 594 \nEDTA, 100 mM NaCl) at 37 °C for 10 min, if not otherwise specified. Proteins were then separated by 595 \n4-20% gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS -PAGE) and 596 \ntransferred onto a nitrocellulose membrane in transfer buffer for 70 min at 200 mA at 4°C. After transfer, 597 \nmembranes were blocked with 4.5% BSA in PBS-T (4 g NaCl, 100 mg KCl, 720 mg Na2HPO4, 123 mg 598 \nKH2PO4, 250 μl Tween -20 in 500 ml) for 60 min at room temperature. After blocking, the membrane 599 \nwas washed using PBS-T once and then incubated overnight at 4°C with the first antibody, Anti-acetyl 600 \nlysine antibody (ICP0380, Immunechem), diluted 1:5000 in PBS -T containing 4.5% BSA. After the 601 \ntreatment with the first antibody, the membrane was washed three times with PBS -T for 10 minutes 602 \neach. Then, it was incubated with goat anti-rabbit IgG-horseradish peroxidase (HRP)linked secondary 603 \nantibody (LF-SA8002, Abfrontier), diluted 1:5000 in PBS -T containing 4.5% BSA, for 60 min. The 604 \nmembrane was washed 4 times with PBS-T for 10 min each time, incubated in Western HRP substrate 605 \n(WBLUF0100, Millipore), and imaged in the Luminograph II (ATTO)70. 606 \n 607 \nIn vitro acetylation assay: DTNB assay 608 \nThe reaction mixture was prepared by adding wild -type or mutant PatZ (1 μM), Acs protein (20 μM), 609 \nDTNB (100 μM, 5,5’-dithio-bis-(2-Nitrobenzoic Acid, Thermo), and AcCoA (from 0 to 20 μM, Cayman) 610 \nin a 200 μL volume of reaction buffer (25 mM sodium phosphate, pH 8.0, 0.5 mM EDTA, 100 mM NaCl). 611 \nGiven that both Acs and AcCoA serve as substrates for PatZ, experiments utilized lower concentrations 612 \nof AcCoA compared to Acs to obtain an accurate initial velocity of CoA release. Absorbance at 412 nm 613 \nwavelength was monitored every 5 seconds for 10 minutes at 37°C using a Flex3 microplate reader 614 \n(Molecular Devices). A standard curve was created using CoA (Avanti) concentrations ranging from 0 615 \nto 100 μM.  Velocity was determined by measuring the consumption of AcCoA using the absorbance 616 \nat 412 nm and then converting this value using the CoA standard curve. This consumption rate was 617 \nthen divided by the time (in minutes) and the amount of PatZs in mg. Velocity and AcCoA concentration 618 \nwere used to fit the wild -type PatZ to a sigmoidal allosteric curve, while the mutants were fitted to a 619 \nMichaelis-Menten curve, using Prism (GraphPad) analytical software, as used in a previous study31. 620 \n 621 \nIsothermal Titration Calorimetry (ITC) assay 622 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nThe protein sample was prepared in the ITC buffer (1X PBS, 100 mM NaCl), and AcCoA was prepared 623 \nin the same ITC buffer at a 15 -fold concentration of each protein. For titration, samples were loaded 624 \ninto a 96-deep-well plate three consecutive wells filled for a single titration: samples for the reaction 625 \ncell, the injection syringe, and the binding buffer for pre -rinsing the reaction cell. All titrations were 626 \nperformed using the automated MicroCal AutoPEAQ ITC instrument (Malvern Panalytical, 627 \nWorcestershire, UK) with 40 injections at 25°C. The experimental data were analyzed using MicroCal 628 \nPEAQ-ITC analysis software with a single-site/double-site binding model. 629 \n 630 \nPhylogenetic analysis 631 \nWe downloaded six predicted structures representing each type of GNAT acetyltransferase from the 632 \nAlphaFold Protein Structure Database53 (AFDB) May 2024 release: P76594, A0A0M8Z1A2, O05581, 633 \nD9SZU3, Q1D7F7 and P0A944 for type I, type II, cAMP-binding type III, ACT type III, NADP+-binding 634 \ntype III and type IV, respectively. Each structure was searched against AFDB50 (AFDB clustered by 635 \n50% 3Di sequence identity) using Foldseek71(version a2f62e) to obtain alignments with at least 90% of 636 \nboth 3Di sequences are covered (foldseek search -c 0.9 --cluster-search 1 --max-seqs 100000). 637 \nResulting alignments were then converted into Kraken2 sample report format and visualized as Sankey 638 \ndiagrams with Pavian72(version 1.0). 639 \n 640 \nWe collected whole genome assemblies of 300 bacterial species from NCBI GenBank 2024 update73,  641 \nincluding Escherichia coli , Streptomyces lividans , Micromonospora aurantiaca , Mycobacterium 642 \ntuberculosis, Myxococcus xanthus and 295 species randomly sampled across the bacterial domain. 643 \nFour archaeal species under genus Methanococcus were included as an outgroup. Maximum likelihood 644 \ntree was then inferred from the concatenation of amino acid alignments of 81 bacterial core genes 645 \nextracted from the assemblies with UBCG74 (version 2.0), applying JTT+F+I+G evolutionary model of 646 \nIQ-TREE75(version 2.2.2.7). 647 \n 648 \nFinally, we’ve put the representative acetyltransferases predicted/experimental structures which has 649 \ndifferent types of regulatory domains: Streptomyces lividans PatA (slPatA, AIJ12884.1) for Type II and 650 \nEscherichia coli YiaC (ecYiaC, UniProt: P37664), while Type III includes Mycobacterium tuberculosis 651 \nPat (MtPat, PDB ID: 4AVB), Micromonospora aurantiaca  PatB ( maPatB, UniProt: D9SZU3) and 652 \nMyxococcus xanthus ActC (mxActC, UniProt: Q1D7F7). slPatA displays similarities to those of Type I 653 \nwith ligands, but the three of Type III GNATs bind to highly specific ligands, including cAMP, Cys/Arg 654 \nand NADP +. Upon cAMP and Cys/Arg, mtPat and maPatB efficiently acetylate their substrates, 655 \nrespectively. But interestingly, mxActC is inhibited when NADP+ is bound. 656 \n  657 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nDATA, MATERIALS, AND SOFTWARE AVAILABILITY 658 \n 659 \nAtomic coordinates of the apo PatZ, liganded PatZ and ATP bound AGD of PatZ models were deposited 660 \nin the RCSB PDB under accession numbers 9SIQ, 9IT0 and 9ISB, respectively. The cryoEM maps 661 \nwere deposited in the EMDB under accession numbers EMD -60849 (apo PatZ) and EMD -60853 662 \n(liganded PatZ). All code used for cryoEM data analysis, structure determination and refinement are 663 \npublicly available. 664 \n 665 \n 666 \nACKNOWLEDGEMENTS 667 \n 668 \nThis work has been supported by the Korean National Research Foundation (2019R1C1C1004598, 669 \n2020R1A5A1018081, 2021M3A9I4021220, 2019M3E5D6063871, 2020R1A6C101A183) and SUHF 670 \nfoundation to S.-H.R and NRF -2018R1A5A1025077, NRF-2019R1A2C2004143, RS-2024-00335765 671 \nto Y.-J. S. The cryoEM data was collected and processed at The Center for Macromolecular and Cell 672 \nImaging (CMCI), Institute for Basic Science (IBS), Global Science experimental Data hub Center 673 \n(GSDC) at Korea Institute of Science facilities. We thank members of CMCI for their discussions and 674 \nsuggestions. We thank members of the Frydman lab for useful discussions and suggestions. We thank 675 \nthe staff scientists for assistance at the beamline 5C of Pohang Light Source. 676 \n 677 \n 678 \nAUTHOR INFORMATION 679 \n 680 \nSchool of Biological Sciences, Institute of Molecular Biology and Genetics, Seoul National University, 681 \nSeoul, 08826, Republic of Korea 682 \nJun Bae Park, Yu-Yeon Han & Soung-Hun Roh 683 \n 684 \nSchool of Biological Sciences and Institute of Microbiology, Seoul National University, Seoul, 08826, 685 \nRepublic of Korea 686 \nGwanwoo Lee, Kyoo Heo & Yeong-Jae Seok 687 \n 688 \nInterdisciplinary Program in Bioinformatics, Seoul National University, Seoul, 08826, Republic of Korea 689 \nDongwook Kim 690 \n 691 \nInterdisciplinary Program in Bioinformatics, School of Biological Sciences, Institute of Molecular Biology 692 \nand Genetics, Artificial Intelligence Institute, Seoul National University, Seoul, 08826, Republic of Korea 693 \nMartin Steinegger 694 \n 695 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nDepartment of Systems Biology, College of Life Science and Biotechnology, Yonsei University, 50 696 \nYonsei-ro, Seoul, 03722, Republic of Korea 697 \nHyun-Soo Cho 698 \n 699 \nDepartment of Chemistry, Chonnam National University, Gwangju, 61186, Republic of Korea 700 \nHyosuk Yun, Chul Won Lee 701 \n 702 \nSchool of Biological Sciences and Institute of Microbiology, Center for RNA Research, Institute for Basic 703 \nScience, Seoul 08826, Republic of Korea 704 \nJeesoo Kim, Juhee Park & Jong-Seo Kim 705 \n 706 \nAuthor Contributions 707 \nS.-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 \nthe experiment. J.B.P., G.L., Y .-Y.H. and K.H. performed molecular cloning, protein expression and 709 \npurification of the proteins. CryoEM sample preparation, data collection and data processing were 710 \ncarried 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 \nrefined the model. For the X -ray crystallography, J.B.P. performed molecular cloning, protein 712 \nexpression, protein purification, crystallization, X -ray diffraction, data processing, model building and 713 \nrefinement under guidance of H .-S.C.. Y.-J.S. and G.L. conducted the enzymatic activity assay and 714 \nbiochemical assay. M.S. and D.K. performed phylogenetic analysis. All authors analyzed and discussed 715 \nthe results. J.B.P., G.L., Y.-Y.H. and D.K. wrote the paper with help from all authors. 716 \n 717 \nCompeting interests 718 \nThe authors declare no competing interest.  719 \n.CC-BY 4.0 International licenseperpetuity. 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It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nFIGURES and LEGENDS 888 \n 889 \n 890 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 891 \nFigure 1. Structure of the apo-PatZ.  892 \na, Domain architecture of bacterial Nε -lysine acetyltransferase that includes the GNAT domain. b, 893 \nComparison of enzymatic acetylation by PatZ and nonenzymatic acetylation by acetyl -phosphate. 894 \nWestern blot analysis using anti-acetylated lysine antibody. CBB denotes coomassie brilliant blue. c, 895 \nAnalysis of the tetrameric state and purity of PatZ using size exclusion chromatography and SDS-PAGE. 896 \nd, CryoEM map and refined model. Each color denotes a distinct subunit. e, The structural arrangement 897 \nof PatZ, consisting of four subdomains. f, The interaction between subunits or subdomains forming the 898 \nlateral interface g, and axial interface. 899 \n  900 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 901 \n 902 \n 903 \nFigure 2. Structure of the liganded-PatZ. 904 \nA, Ligand bindings to tetrameric PatZ and their binding sites. Four ATPs were docked into the tetramer 905 \nstructure based on the ATP bound AGD crystal structure.  B, PatZ subdomain schematic and its ligand 906 \nbinding site. 2Fo-Fc electron density map of ATP and cryoEM maps of two AcCoAs and phosphate. C, 907 \nMolecular interactions between PatZ GNATD and AcCoA.  D, Molecular interactions between the axial 908 \ndimer interface and phosphate, and between PatZ CBD and AcCoA.  E, Interaction between PatZ AGD 909 \nand ATP. *’w’ denotes water molecule. Hydrogen bonds and electrostatic interactions are represented 910 \nby black dashed lines. 911 \n 912 \n 913 \n914 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 915 \nFigure 3. AcCoA-induced conformational change and catalytic residues in GNATD.  916 \nA, Structural rearrangement of GNAT (red cartoon). Substrate gating motifs in the apo -state and 917 \nAcCoA-bound state are illustrated in blue color and purple color, respectively. AcCoA is shown in ball-918 \nand-stick style. B, Molecular interaction between AcCoA and PatZ GNATD. Magenta color is part of the 919 \nsubstrate gating motif of GNATD. C, Structural influence of the AcCoA -induced movement of gating 920 \nmotif on the formation of the substrate binding site. D, Electrostatic surface charge and amino acid 921 \nresidues comprising the acceptor lysine binding site in GNATD. E, Comparison of relative functional 922 \nlevel between wild-type and point mutants which comprise acceptor lysine binding pocket. Western blot 923 \nanalysis was performed using an anti-acetylated lysine antibody. F, Homology structure based catalytic 924 \nmechanism prediction of PatZ using slPatA and seAcs complex structure. Hydrogen bonds are 925 \nrepresented by black dashed lines. G, Proposed acetyltransferase mechanism of PatZ. 926 \n 927 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 928 \n  929 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nFigure 4. Allosteric coupling of regulatory domain to GNATD.  930 \nA, Schematic of domain truncation construct design and its size exclusion chromatography peak pattern. 931 \n‘tr’ denotes truncation. B, Comparative enzymatic activity analysis of subdomain truncated constructs 932 \nvia western blot. Western blot analysis was performed using an anti-acetylated lysine antibody c, ITC 933 \nprofiles of AcCoA binding to wild -type, truncated forms and RR (R26E/R81E) mutant of PatZ.  D, 934 \nComparative enzymatic activity analysis of wild-type, RR mutant and GNAT-only constructs of PatZ.  E, 935 \nStructural variability comparison between apo - (left) and liganded -PatZ (right).  F, Comparison of 936 \nbackbone RMSD between apo - and liganded-PatZ (purple) and backbone RMSD within apo -PatZ 937 \nvariability (red) and liganded -PatZ variability (blue). Asterisk refers to the substrate gating motif of 938 \nGNATD. G, Ligand binding-mediated structural change of PatZ regulatory domain h, Structural effect 939 \nof AcCoA binding on the interaction between GNATD and regulatory domain. Hydrogen bonds are 940 \nrepresented by dashed lines. I, Role of substrate gating motif in changes in interaction between GNATD 941 \nand regulatory domain following AcCoA binding  j, Substrate access channel formed by AcCoA -942 \nmediated GNATD conformational change and the role of substrate gating motif. Magenta color is a 943 \nspace filling model of substrate gating motif. 944 \n  945 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 946 \n 947 \n 948 \nFigure 5. The regulatory domain of PatZ contributes to the formation of Acs binding pocket.  949 \na, Predicted structure of the E.coli PatZ-E.coli Acs complex using AlphaFold 41. Each domain is 950 \nrepresented in a different color using cartoon -style representation. Acs K609 is represented in stick 951 \nstyle. b, Electrostatic surface potentials of Acs and PatZ. Red and blue for negative and positive charges, 952 \nrespectively, and white color represents the neutral area. The circularly linked different parts represent 953 \nthe interacting components. c, Acs-CTD and PatZ-LCD interaction (left panel) and Acs-CTD and PatZ-954 \nCBD interaction (right panel). The circled regions denote the positions of the labeled residues. d, Time-955 \ndependent comparison of PatZ mutant activity relative to wild -type. Western blot analysis using anti -956 \nacetylated lysine antibody (left panel) and normalized activity comparison graph (right panel). 957 \n 958 \n 959 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 960 \n 961 \nFigure 6. Phylogenetic analysis and catalytic mechanism  962 \na, Schematic illustration of ligand -induced domain-specific conformational changes regulating PatZ 963 \ncatalytic activation. b, Visualization of Type I, Type II, cAMP -binding type III, ACT type III, NADP+ -964 \nbinding Type III and Type IV c, Venn diagram showing the overlap of GNAT types. Each number 965 \nrepresents the count of bacterial species containing the corresponding GNAT type. 966 \n 967 \n 968 \n 969 \n 970 \n971 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nEXTENDED DATA and LEGENDS  972 \n 973 \n 974 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 975 \nExtended Data Figure 1. CryoEM processing workflow of apo -state PatZ. a, Schematic of data 976 \nprocessing pipeline with representative cryogenic electron microscopy micrographs and two -977 \ndimensional averages. b, Angular distribution of views in the 3D reconstruction (left view and right view, 978 \nrotated by 90˚). c, Gold-standard fourier shell correlation (GSFSC) curve. d, Map in front view, colored 979 \naccording to local resolution. e, CryoEM density map features corresponding to the four subdomains. 980 \nf, Side chain Q-score distribution across the four domains. 981 \n 982 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 983 \n  984 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nExtended Data Figure 2. CryoEM processing workflow of liganded-PatZ and 2Fo-Fc electron density 985 \nmap of ATP in ATP bound AGD crystal structure. a, Schematic of data processing pipeline with 986 \nrepresentative cryogenic electron microscopy micrographs and two-dimensional averages. b, Angular 987 \ndistribution of views in the 3D reconstruction (left view and right view, rotated by 90˚). c, Gold-standard 988 \nfourier shell correlation (GSFSC) curve. d, Map in front view, colored according to local resolution.  e, 989 \nCryoEM density map features corresponding to the four subdomains.  f, Side chain Q-score distribution 990 \nacross the four subdomains.  g, Model refinement results for two AGD chains located in a single 991 \nasymmetric unit of the crystal, with 2Fo-Fc electron density map of ATP contoured at 1σ. 992 \n  993 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 994 \n 995 \nExtended Data Figure 3. Structural similarity between GNATD of ecPatZ and slPatA, and sequence 996 \nconservation level of PatZ E809. a, Structural similarities between ecPatZ GNATD and slPatA GNATD. 997 \nb, ConSurf analysis of sequence conservation for PatZ E809. 998 \n 999 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 1000 \nExtended Data Figure 4.  a Comparison of relative functional level between wild-type and truncated 1001 \nmutants. Asterisk mark refers to the truncated PatZ s. b, Structural comparison of three distinct 1002 \nconformational states (Classes I -III) of apo -state PatZ LCD identified through 3D classification . c, 1003 \nQuantitative comparison of conformational changes in the regulatory domain induced by ligand binding. 1004 \n 1005 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 1006 \n 1007 \nExtended Data Figure 5. Prediction of PatZ-Acs complex structure using AlphaFold3. a, The Predicted 1008 \nAligned Error (PAE) matrices calculated via AlphaFold3. b, pLDDT for all residues belonging to the Acs 1009 \nand PatZ. c, 3D visualization of prediction error estimation using PAE plot focused on PatZ -Acs 1010 \ninteraction part. Stick style refers to residues originating from two chains within a 4 Å distance. The 1011 \ncoloration follows the pLDDT color spectrum. 1012 \n 1013 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 1014 \nExtended Data Figure 6. The PAE matrices that are calculated via AlphaFold3. a, E.coli YiaC (Type 1015 \nIV). b, S. lividans PatA (Type II). c, M. aurantiaca PatB (Type III). d, M. xanthus ActC (Type III). e, 1016 \nDomain-wide distribution of GNAT acetyltransferase types across a phylogenetic tree of 300 bacterial 1017 \nspecies.  Different types are indicated by dots in the concentric circles, where a filled dot indicates the 1018 \nspecies containing a structure similar to the representative structure of each type. For each type, 1019 \npercentage of the species abundance among the bacterial domain as well as the representative 1020 \nstructure are displayed on the location of corresponding species. f, Distribution of GNAT types across 1021 \nthe bacterial domain displayed as Sankey plots. Shown are the ranks of domain (D) and phylum (P), 1022 \nwith the top ten most abundant groups per rank; numbers indicate the amount of underlying species. 1023 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 1024 \n 1025 \n 1026 \nExtended Data Figure 7. Structural similarity of gating-motif-containing helices. Comparative analysis 1027 \nof sequence-structure similarity in GNAT gating motifs across eukaryotes and prokaryotes. Distinct 1028 \nhelices containing the gating motif are differentiated by color. Similarities among the sequences are 1029 \ngiven a gray background. 1030 \n  1031 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\n 1032 \nExtended Data Figure 8. Western blot analysis using anti -acetylated lysine antibody for comparison 1033 \nof acetylation by type I E. coli PatZ and type IV E. coli YiaC. 1034 \n  1035 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nSupplementary Table 1. CryoEM data collection, refinement and validation statistics 1036 \n 1037 \n  1038 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nSupplementary Table 2. X-ray crystallographic data collection and refinement statistics 1039 \n 1040 \n 1041 \nSupplementary video 1. Tetramer assembly and subdomain composition of PatZ 1042 \n 1043 \nSupplementary video 2. Ligands that bind to PatZ and their binding sites 1044 \n 1045 \nSupplementary video 3. Ligand-mediated conformational change of PatZ GNAT domain 1046 \n 1047 \nSupplementary video 4. Ligand-mediated global conformational change of PatZ 1048 \n 1049 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nN\nC\nAbs (280nm)\n    0         5        10       15        20       25 (mL)\n(kDa)\n150\n100\n75\n670 158 44 17 (kDa)\nca b\nGNATDLCD\n1 451 709 886285\nN CAGD\nCatalytic\ndomainRegulatory domain\nCBD\n90˚\n180˚\n90˚\n1\n2 3\n4\n1 2\n3 4\n1 2\n3 4\nCoA-binding \ndomain (CBD)\nLigase-CoA \ndomain (LCD)\nATP-grasp \ndomain \n(AGD)\nGNAT \ndomain \n(GNATD)\nN\nC CBD\nLCD\nGNATD\nAGD\nCBD\nLCD\nGNATD\nAGD\nCatalytic\ndomain\nRegulatory \ndomain\n1 2\n3 4\n1 2\n3 4\n1 2\nLateral \ninterface\nAxial \ninterface\n2\n4\nCBD\nLCD\n90˚\nCBD\nd e\nf g\n1\n2\n2\n1\nK171\nQ169\nE408\nH409S410\n2\n4\nA53\nA53\nG56\nG56\nP43 V44\nP43V44\nRegulatory domain GNAT domain\nDomain configuration\nType I\nType II\nType III\nType IV\nType V Acs\nAnti-AcLys\nCBB staining\nAcs\nPatZ + + + + - - - - -\n1 mM AcCoA - + + + - - - - -\n1 mM AcP\n-\n- - -\n+\n+ + + -\nAcs + + + + + + + + +\n0m 10m 1h 16h 0m 10m 1h 16h 16h\n10 mM AcP\n-\n- - -\n-\n- - - +\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nATP\nAcCoA\nAcCoA\nPhosphate\n1 2\n3\n4\n1 2\n3 4\nCBD LCD\nAGD\nGNATD\nATP\nAcCoA\nATP\nAcCoA\nAcCoA\nAcCoA AcCoA\nPhosphate\nA299\nA298\nR343\nA158\nA157\nS156\nphosphate\nAcCoA\nLCD-CBD interface\nK860\nR825\nG824\nL823\nK189G822\nL821\nw\nV814\nL813\nV812\nN851\nF810\nGNATD\nAcCoA\nL813\nV814\nN851\nK860\nR825\nA577\nQ574\nK523\nR579\nE584\nH676\nH531\nK532S533\nAGD\nK523Q574\nR579\nE584\nH676\nH531\nATP\na b\nc\nW51\nT48\nS21 R26\nN78\nR81\nAcCoA\nK23\nCBD\nR81\nN78\nR26\nK23S21W51\nT48\nA299\nA298\nA158\nA157\nS156 w\nw\nR343\nd\ne\nRegulatory module\nAcCoA\nbinding\nAcCoA\nbinding\nATP\nbinding\nphosphate\nbinding\nGNATDLCD\n1 451 709 886285\nN AGDCBD\nCatalytic\nmodule\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nSubstrate \nbinding\nsite\nAcCoA\nbinding\nsite\napo-\nstate\nAcCoA-\nstate\nAcCoA \nbinding\n D750\nY753\nM848\nN851\nAcCoA\nAcCoA \nbinding\na\nF756\nR754\nI846\nE809\nAcCoA\nf\nE809\nF810\nI846\nAcCoA\nE123\nE160\nK609\nV124\nH2O\nslPatA\nGNATD \nseAcs\nCTD (4U5Y):\nAcCoA\nI846\nF810\nE809\nH2O\nK609\necPatZ\nGNATD \nseAcs\nCTD (Superposition):\ng\nb\nWB\nCBB Acs\nAcs\n+ + + + + +\n- + + + + +\n+ + + + + +PatZ\nAcCoA\nAcs\nWT WT R754A F756A E809A I846A\nO\n- O\nGlu809\nCoA-S\nO\nPatZ\nLysine-substrate\nO\nHO\nGlu809\nPatZ\nAcLys-substrate\nCoA-SH\nCoA-SH\nAcCoA\nH\nH O\nLysH\n+..\nNH2\nH\nH O\nAcLys\nNH\nO\nPatZ-GNATD\nLys-substrate\nPatZ-GNATD\nLys-substrate\nd e\nc\nSubstrate-\ngating motif\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\na\nNTD CTD\nGNATD\nK609\nLCD\nR395\nY398\nLCD\nCTD\nE570\nK228\nN227\nCBD\nCTD\nD634\nWB\nCBB \nAc-CoA\nAcs\n+ + -\n+ + + +\n+ + - +\n+ + + +\n+\n+\n10 30 10 30 10 30\nPatZ WT PatZ R395A,Y398A PatZ N227A,K228A\n(min)\n-\n0 0 0\nR395A,\nY398A\nN227A,\nK228A\n0\n0.5\n1.0\n1.5\nPatZ\nWT\nNomalized activity\n*\n**\nNTD CTD\nGNATDK609\nLCD\nCBD\nPatZAcs\nCBD\nAGD\nPatZ\nAcs\nb\nc d (i) (ii)\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\ne\nh i\ng\nj\nc\nf\nd\n0 20 40 60 80 100 120\n1\n0\n-1\n-2\n-3\n-4\n0 1 2 3\n0\n-10\n-20\n-30\nDP (μM)\nRatio\nTime (min)\nWT\nKd1 0.60 μM\nKd2 3.98 μM\n0 20 40 60 80 100 120\nTime (min)\n0\n-10\n-20\n-30\nΔH (kJ/mol)\n1\n0\n-1\n-2\n-3\n-4\n0 1 2 3Ratio\nRD-only\nKd 5.15 μM\n0 1 2 3Ratio\n1\n0\n-1\n-2\n-3\n-4 DP (μM)\n0\n-10\n-20\n-30\n0 20 40 60 80 100 120\nR26E, R81E\nKd 12.10 μM\n0 20 40 60 80 100 120\n0\n-5\n-10\n-25\nΔH (kJ/mol)\n-15\n-20\n1\n0\n-1\n-2\n-3\n-4\n0 1 2 3Ratio\nGNATD-only\nKd 8.13 μM\n0\n20\n40\n60\n5 10 15\nAcCoA (μM)\nVelocity (μM/min/mg)\n0\nWT\nR26E, R81E\nGNATD-only\nTime (min) Time (min)\n    0           5            10         15         20          25(mL)\nAbs (280nm)\n670 158 44 17 (kDa)\nWT\nVoid\nWT\ntrCBD-LCD\nRD-only\nGNATD-only\n1 451 709 886285 461\nAGDLCD GNATN CCBD\nAGD GNATN C\nGNATN C\nAGDLCDN CCBD\na\ntrCBD-LCD\n- - + + + + +\n- + - + + + +\n+ + + + + + +\nPatZ\nAcCoA\nAcs\nWB\nWT WT GNATD-onlyRD-only\nAcs\nAcs\nCBB staining\nLCD GNATN CCBD\ntrAGD\nGNATD-only\ntrCBD-LCD\ntrAGD RD-only\nb\nVariability in apo-PatZ Variability in liganded-PatZ\nRMSD RMSD\n0 Å\n20 Å\n0 Å\n20 Å\nRMSD (Å)\nGNATDLCD AGDCBD\n0\n10\n20\n30\n0 200 400 600 800\n0\n5\n10\n15\n*apo- vs liganded-PatZ \napo-PatZ \nliganded-PatZ \nR343\nS156\nR343\nS156\nR343\nS156\nR343\nS156\nPhosphate + AcCoA\n1 2\n3 4\n1\n21\n2\n1\n21\n2\nAcCoA\nAcCoA\nOverall B-factor = 77.6\nInterface area (Ų) = 1002.7\nOverall B-factor = 39.0\nInterface area (Ų) = 2094.8\nRelaxed structure Tense structureRMSD=8.1 Å\n37°\n40°\nAcCoA \nY753\nR197 Y194\nE7F756\nN-term\nE736\nN769\nQ772\nT452\nR450\nC\nSubstrate \naccess\nAcCoA\nCBD\nAGD\nGNATD\nGNATD\nCBD\nAGD\nCBD\nGNATD\nCBD\nGNATD\nAcCoA \nY753\nY753\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint \n\nCoAAc\nGNATD\nAGD\nCBD\nLCD\nGating-\nmotif\nAcs\nSubstrate\na\nNADP+\ncAMP\n Cys/Arg\nAcCoAATP PO-\n4\n AcCoA ATP\nRegulatory GNAT \nType I Type II\nType III Type IV\n348\n112\n67\n3,582\n14,343\n1,543\n865\n317\n7 7\n93\nType I\nType II\nType III\nType IV\nCoA\nInhibited conformation\nGating motif: closed  \nSubstrate access channel : closed\nActivated conformation\nGating motif: open \nSubstrate access channel : open\nAcetyl-Acs\nSubstrate\nSubstrate \naccess\nTethered_down\nposition\nUp \nposition\nGating-\nmotif\nSubstrate \naccess Binding\nsite\nGNATD\nAGD\nCBD\nLCD\nb c\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted November 13, 2024. ; https://doi.org/10.1101/2024.11.12.623305doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}