The importance of helical structures to the overall activity and structural stability of a lipase from Pseudomonas aeruginosa PAO1 in n-hexane.

Abstract

Bacterial lipases are versatile extracellular enzymes with a catalytic triad at the active site and a flexible 'lid' that modulates catalytic accessibility. We combined computational modeling with preliminary in vitro testing to assess the structural stability and activity of the Pseudomonas aeruginosa PAO1 lipase (PAL). We evaluated several systems consisting of the native and mutant forms of the lipase in n-hexane using molecular dynamics simulations. Structural stability was assessed by calculating the B-factor for each system. We measured the gorge radius of the catalytic channel and the RMSD of the catalytic triad to approximate enzymatic activity. Based on the correlation of these metrics, mutant forms were selected for their potential activity and stability. Selected mutant forms were expressed in E. coli BL21, mass-produced, and validated through a lipase-catalyzed esterification assay in n-hexane. Several helices outside the 'lid' region were found to influence lid conformational switching. Moreover, our preliminary experimental results show promise in validating our in silico predictions. Our integrated in silico and in vitro pipeline offers a promising approach for designing and producing industrially relevant lipases.