Conformational gating in ammonia lyases

Abstract

Ammonia lyases (AL) are enzymes of industrial and biomedical interest. Knowledge of AL structure-dynamics-function relationship would be instrumental for making use of the application potential of these enzymes. We investigated, using microsecond molecular dynamics, 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 {beta}5-2 loop, and the helix-hairpin-loop subdomain. We showed that they undergo conformational changes switching from occluded to open states. We observed that these rearrangements are coupled to changes in the accessibility of the active site. The {beta}5-2 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 {beta}5-2 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 protein engineering and therapeutic purposes.