(A) View from an MBLAC1 crystal structure (PDB ID: 4V0H) showing secondary structure elements and the di-metal containing active site. Helices are in cyan, β-strands in yellow, and metal ions are orange spheres. Dashed line (in gray) indicates unrefined residues (aa 51–66). (B) View of metal binding and active site residues with a representative electron density map (3.0 σ mFo-DFc OMIT; cyan mesh) for the sidechains of His116 (Nε2 to M1: 2.33 Å), His118 (Nε1 to M1: 2.35 Å), Asp120 (Oδ2 to M2: 2.9 Å), His121 (Nε2 to M2: 2.37 Å), His196 (Nε2 to M1: 2.27 Å), Asp221 (Oδ2 to M1: 2.14 Å; Oδ2 to M2: 2.12 Å), His263 (Nε2 to M2: 2.06 Å) and the two water molecules (red spheres) which coordinate (black dashed lines) to the metals (orange spheres). The BBL MBL numbering system is used (Galleni et al., 2001) (active site motif numbers in parentheses) (Pettinati et al., 2016). Numbering as used in our PDB deposited structure is in bold. (C) Inductively coupled plasma mass spectrometry (ICP-MS) experiments reveal that in human cells MBLAC1 binds zinc. (D) MBLAC1 dimer model calculated by PISA (Krissinel and Henrick, 2007). A surface representation is shown for protomer A/C. (E) Multi-angle laser light scattering (MALS) analyses of E. coli produced wt and active site variant MBLAC1, and of HEK293 produced wtMBLAC1. Peak A likely represents a trimer (~80000 Da); peak B (~54000 Da) a dimer; and peak C (~27000 Da) a monomer. wt MBLAC1 is predominantly dimeric, the active site variant is mainly monomeric. The latter observation correlates with loss of activity of active site variant MBLAC1 (main text Figure 4a), the proposal that the dimer is catalytically active, and the observation that the active site is close to the dimer interface. MALS experiments were carried out at the Biophysical Services of the Biochemistry Dept., Oxford University. (F) Non-denaturing PAGE analyses indicates wt MBLAC1 produced in either E. coli or HEK293 cells is predominantly dimeric; a higher oligomeric state (tetramer) is also observed for the bacterial produced wt protein. The active site substituted enzyme produced in E. coli shows loss of oligomerization. wtMBLAC1 produced in either, E. coli or HEK293 cells shows the same oligomerization behavior. (G) Non-denaturing electrospray ionization mass spectrometry deconvoluted spectra of recombinant MBLAC1 produced in E. coli indicates that MBLAC1 is dimeric, binding two metal ions. Peak A (54880 Da) represents the dimer with two divalent transition metal ions (zinc or iron) bound to each monomer (+224 Da); peaks B and C (27440 and 54880 Da, respectively) correspond to monomer and dimer without bound metal, following metal removal using EDTA. (H) Superimposition of the MBLAC1 (cyan; metals in orange) and LACTB2 folds (PDB ID: 4AD9) (Levy et al., 2016) (pink; metals in grey) reveals a high degree of overall similarity (RMSD 2.2 Å over 153 Cα atoms).