Fig. 1: The ATP and FAD-binding sites in the ACOX-1.1 crystal structure.

a The crystal structure of the ACOX-1.1(E434A) homodimer (PDB: 5K3I) shown as both a cartoon and surface representation with subunit A in orange and subunit B in cyan. The two ATP molecules are boxed and labeled, while the two FAD molecules are only labeled at their corresponding binding sites. Both of the FAD and ATP-binding sites are at the interface between the two subunits in the dimer. However, for clarity in this manuscript, we refer to one of the FAD/ATP as being predominantly associated with subunit A (given that more of the binding site residues are associated with subunit A) and one of the FAD/ATP as being predominantly associated with subunit B. b A cartoon representation of ACOX-1.1(E434A) (PDB: 5K3I), with bound FAD and ATP, overlaid with that of ACOX-1.1 ApoForm I (PDB: 5K3G) in white. The FAD/ATP-binding sites that are shown are predominantly associated with subunit A. Residues, which when mutated result in a lack of retention of both FAD and ATP to purified ACOX-1.1, are highlighted, and they include W189, Q340, K391, N437, H396, and R536¹⁵. The subunit that they are part of is indicated with a superscript. In the holo ACOX-1.1(E434A) structure, K391 hydrogen bonds to main-chain carbonyls in the catalytic loop (which forms a hydrophobic interaction surface for FAD) and to N437 (which in turn hydrogen bonds to ATP). In the apo ACOX-1.1 structure, the catalytic loop, K391, and N437 are disordered, and H396 is in a different position than it is in holo ACOX-1.1(E434A). The catalytic residue, E434, is not depicted in the catalytic loop in this figure because it is mutated to Ala in the holo ACOX-1.1(E434A) structure, and it is disordered in the apo ACOX-1.1 structure.