A Dimer between Monomers and Hexamers – Oligomeric Variations in Glucosamine 62 Phosphate Deaminase Family.

: In deaminase (NagB) catalyzes the last step in the conversion of sialic acid into Fructose-6-27 phosphate, which enters the glycolytic pathway. The enzyme exists as a hexamer in Gram-28 negative bacteria and is shown to be allosterically regulated. In Gram-positive bacteria, it 29 exists as a monomer and lacks allosteric regulation. Our identification of a dimeric Gram-30 negative bacterial NagB motivated us to characterize the structural basis of the various 31 oligomeric forms. We characterized the crystal structures of NagB from two Gram-negative 32 pathogens, Haemophilus influenzae (Hi) and Pasturella multocida (Pm) . The Hi -NagB is 33 active as a hexamer, while Pm -NagB is active as a dimer. We confirm that this is not a 34 crystallographic artifact by cryo-electron microscopy. Both Hi-NagB and Pm-NagB contain 35 the C-terminal helix, and the residues in the interface involved in oligomerization are 36 conserved. The hexamer is described as a dimer of trimers. In the Pm-NagB dimer, the 37 dimeric interface is conserved. This would suggest that the three possible oligomeric forms of 38 NagB are a monomer, a dimer, and a trimer of dimers. Computational modeling and MD 39 simulations indicate that the residues at the trimeric interface have less stabilizing energy of 40 oligomer formation than those in the dimer interface. We propose that Pm-NagB is the 41 evolutionary link between the monomer and the hexamer forms.

97 Diffraction data were collected to 2.3 Å resolution from a single crystal of PmNagB and 3.0 Å 98 resolution from a single crystal of HiNagB on the PROXIMA-1 beamline at the SOLEIL 99 synchrotron source. Data were processed with XDS/XSCALE[13] and scaled with AIMLESS 100 from the CCP4 suite [14]. The data-processing statistics are provided in Table 1.

Data Set HiNagB PmNagB
Data processing Space group a, b, c (Å)  . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. 107 automatically added during the process of refinement but were manually checked for hydrogen 108 bonding and density fit to both (2|Fo|-|Fc| ) and (|Fo| -|Fc|) maps. The structure-solution and 109 refinement statistics are provided in Table 1.

2.4 | Electron cryomicroscopy
111 Protein was purified as described for crystallography. 3 µl of HiNagB at 1 mg/ml were applied 112 to Quantifoil holey carbon grids (R 1.2/1.3, Au 300 mesh) with blotting and freezing 113 accomplished on a Vitrobot mark IV at 18°C and 100% RH. The images of these grids showed 114 nice isolated particles of NagB. The data of NagB were collected with Titan Krios and Falcon 115 3 detector in counting mode at 1.07 Å/pixel sampling with images exposed for 60 seconds with 116 a total accumulated dose of ~30 e -/Å 2 and dose fractionated into 25 frames, with each frame 117 having a dose of ~1.2 e -/Å 2 . An algorithm within Relion3 was used for full-frame alignment 118 and dose weighting [18]. The summed images were then used for automated particle picking 119 with Gautomatch (Kai Zhang) and templates derived from manual picking, and CTF was 120 estimated with Gift [19]. Particles were extracted with a box size of 180 pixels, and 2D 121 classification was performed. The projections of the PDB structure were created using EMAN2 122 software [20]. The PDB file from the X-ray data was converted to a map file with a box size 123 of 180 pixels and a pixel size of 1.07 Å. The map was low pass filtered to 10 Å, and the 2D 124 projections were created for comparison.
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. 132 an orthorhombic box shape was used with a buffer distance of 10 Å, followed by a 133 minimization of the box size. The system was neutralized, and salt (NaCl) was added. The  (Table 1). The PDB ID assigned for PmNagB is 7LQN.  (Table 1). The PDB ID assigned for PmNagB is 7LQM.  . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. 188 To ensure that the purified proteins with quaternary structures were active deaminases, we 189 conducted steady-state kinetic studies to check the activity of the enzymes. We used ammonia   . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022.   . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. 223 First, we analyzed the trimeric interface and observed that the interface stabilizing energy for 224 HiNagB and E.coliNagB was -100 kJ/mol and -170 kJ/mol, respectively. However, the 225 calculated trimeric interface stabilizing energy for PmNagBHexa* was only around -33 kJ/mol.  . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. ; https://doi.org/10.1101/2022.07.06.499027 doi: bioRxiv preprint 231 To investigate the crucial role of these two residues on trimer formation, we performed in-232 silico mutations of these two residues, E164Q-Q210L, in the trimeric interface of the 233 PmNagBHexa*, to those found in HiNagB followed by minimization (100 sd step), and 234 calculated the interface energy. The results suggested that the mutations did not yield any 235 significant difference in the stabilization energies. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. ; https://doi.org/10.1101/2022.07.06.499027 doi: bioRxiv preprint 252 relationship is not just a pure rotation from A to B, but there is also a translation of 0.38Å along 253 the rotation axis. In the case of A to C, the translation vector is 0.14Å, and in the case of B to 254 C, it is 0.54Å. These are hence not just rotations but also include a translation along the 255 axis. This should result in slight asymmetry in the interactions between the A, B, and C 256 subunits. These are minor differences and yet are clearly visible in the attached dimplot 257 ( Figure 6). This asymmetry is not conserved among the four different trimers in the 258 asymmetric unit. One could say that while the protein's oligomeric state is hexameric, there is 259 some shear between the subunits. It is currently difficult to say if this shear has an impact on   . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. ; https://doi.org/10.1101/2022.07.06.499027 doi: bioRxiv preprint 297 298 The authors declare that they do not have any competing interests.

299
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. ; https://doi.org/10.1101/2022.07.06.499027 doi: bioRxiv preprint 300 301 . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 6, 2022. ; https://doi.org/10.1101/2022.07.06.499027 doi: bioRxiv preprint