Conformational gating in ammonia lyases

Ammonia lyases (AL) are enzymes of industrial and biomedical interest. To succeed in AL biotherapeutic applications, the knowledge of their structure-dynamics-function relationship would be beneficial. We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. In particular, we identified two regulatory elements in the MAL structure, i.e., the β5-α2 loop, and the helix-hairpin-loop subdomain. We showed that they undergo conformational changes switching from 9occluded9 to 9open9 states. We observed that these rearrangements are coupled to changes in the accessibility of the active site. The β5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of tunnels from the protein surface to the substrate binding site, making the active site more accessible to the substrate when they are in an open state. We pinpointed a sequential mechanism, in which the helix-hairpin-loop subdomain needs to break a subset of intramolecular interactions first, to then allow the opening of the β5-α2 loop and, as a consequence, make the AL catalytic pocket accessible for the substrate. Our data suggest that protein dynamics need to be considered in the design of new AL variants for engineering and therapeutic purposes.