Conformational gating in ammonia lyases

Abstract

14Ammonia lyases (AL) are enzymes of industrial and biomedical interest. Knowledge 15 of AL structure-dynamics-function relationship would be instrumental for making use 16 of the application potential of these enzymes. We investigated, using microsecond 17 molecular dynamics, the conformational changes in the proximity of the catalytic 18 pocket of a 3-methylaspartate ammonia lyase (MAL) as a model system. In 19 particular, we identified two regulatory elements in the MAL structure, i.e., the β 5-α2 20 loop, and the helix-hairpin-loop subdomain. We showed that they undergo 21 conformational changes switching from 'occluded' to 'open' states. We observed that 22 these rearrangements are coupled to changes in the accessibility of the active site. 23 The β 5-α2 loop and the helix-hairpin-loop subdomain modulate the formation of 24 tunnels from the protein surface to the substrate binding site, making the active site 25 more accessible to the substrate when they are in an open state. We pinpointed a 26 sequential mechanism, in which the helix-hairpin-loop subdomain needs to break a 27 subset of intramolecular interactions first, to then allow the opening of the β 5-α2 loop 28 and, as a consequence, make the AL catalytic pocket accessible for the substrate.29 2 Our data suggest that protein dynamics need to be considered in the design of new 1 AL variants for protein engineering and therapeutic purposes. 2 Keywords 3 molecular dynamics simulations, conformational changes, disordered regions, 4 conformational switches, correlated motions 5 6

Structure-guided protein engineering of ammonia lyase for efficient synthesis of sterically bulky unnatural amino acids

“…The Ec MAL homology model was created using SWISS-MODEL ( https://swissmodel.expasy.org/ ), taking MAL from Clostridium tetanomorphum (PDB ID:1KKR) as a template (Lambrughi et al 2019 ). The modeled protein structure was evaluated through a Ramachandran plot, and the percentage of residues in the allowed region was 98.6%.…”