Elatico_2020_J.Mol.Graph.Model_100_107657

Lipases are important enzymes in many biochemical industries, thus making them attractive targets for protein engineering to improve enzymatic properties. In this work, a ''reverse engineering'' approach was explored: disrupt secondary structures to determine their contribution to enzyme stability and activity. All the alpha-helices of the lipase from Pseudomonas aeruginosa PAO1 (PAL) were systematically disrupted using computational proline mutagenesis and molecular dynamics (MD) simulations. This method identified the alpha3 mutant (R89P), located within the vicinity of the active site, to be significantly important for stability and activity. In addition, the alpha6 system (L159P), part of the ''cap'' domain that regulates substrate entry into the active site, was found to be critical for activity as it pushed the lipase to adopt a completely closed conformation. The perturbation introduced by the proline mutations resulted in increased backbone flexibility that significantly decreased protein stability. Moreover, mutations within the cap domain helices - alpha4 (A115P), alpha5 (S132P, G139P), alpha6 (L159P), and alpha7 (R169P) - resulted in increased flexibility of the N-terminal region of the alpha5 helix, the mobile ''lid'' helix, that pushes the gorge into a partially closed conformation. The alpha6 mutation (L159P) further increased the flexibility of the helix-loop region at the C-terminal end of the alpha5 helix to push the lid into the fully closed state. Therefore, the alpha3 and alpha6 helices could be ''hot spots'' for stabilizing mutations that could improve the overall enzyme stability and activity this lipase. The insights obtained in this work may be validated experimentally in future works.