Computational reverse protein engineering of a lipase from Pseudomonas aeruginosa in n-hexane

Abstract

This study used computational methodologies to investigate the behavior of Pseudomonas aeruginosa PAO1 lipase (PAL) in n-hexane. Molecular dynamics simulations were employed to evaluate perturbations on the structural stability of the secondary structures of the lipase. B-factor analysis served as an indirect stability measure, while gorge radii indicated enzymatic activity. Findings reveal that the α3 helix significantly influences the structural stability of PAL, though stability-activity relationships remain complex. Mutant forms characterized by dual-lids — including mutants of 310−2, α6, and the triple-point mutant of the eight β-strands — exhibited highly open gorges throughout simulations, with flexibility in typically rigid residues of non-310 lid1 helices and highly flexible dual-lid 310 helices. Perturbation of lid 310−2, especially prominent in the α8 mutant, suggests a potential trigger mechanism, as its gorge radii fluctuated considerably between open and closed states. Other significant regions include the α2 helix and the α7–α8 loop, which displayed substantial flexibility in both highly open and closed systems, indicating their roles as hinges in the dual-lid configuration. Most mutant forms tended to remain open, with only three mutants — α7, α1, and α2 — remaining rigidly closed, supporting reports that lipases prefer an open state in organic media. For potential enhancement, the proline mutant of 310−2 (R119P) emerged as the most promising, balancing stability and dual-lid flexibility. Collectively, these findings offer insights into structural dynamics of PAL, elucidate its gating mechanism, and identify strategies for enhancing stability, with implications for biotechnological applications.