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Background Ammonia lyases are enzymes of industrial and biomedical interest. Knowledge of structure-dynamics-function relationship in ammonia lyases is instrumental for exploiting the potential of these enzymes in industrial or biomedical applications. Methods We investigated the conformational changes in the proximity of the catalytic pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. At this scope, we used microsecond all-atom molecular dynamics simulations, analyzed with dimensionality reduction techniques, as well as in terms of contact networks and correlated motions. Results We identify two regulatory elements in the MAL structure, i.e., the β5-α2 loop and the helix-hairpin-loop subdomain. These regulatory elements undergo conformational changes switching from ‘occluded’ to ‘open’ states. The 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 catalytic site, making the active site more accessible to the substrate when they are in an open state. Conclusions Our work pinpoints a sequential mechanism, in which the helix-hairpin-loop subdomain of MAL needs to break a subset of intramolecular interactions first to favor the displacement of the β5-α2 loop. The coupled conformational changes of these two elements contribute to modulate the accessibility of the catalytic site. General significance Similar molecular mechanisms can have broad relevance in other ammonia lyases with similar regulatory loops. Our results also imply that it is important to account for protein dynamics in the design of variants of ammonia lyases for industrial and biomedical applications.