Propeptide in Lipase: New Insights into Its Mechanism of Activity and Substrate Selectivity by Computational Design.

Most fungal lipases contain a propeptide, which is very important for their function and substrate selectivity. In the present study, lipase (RCL) was used as a research model to explore the mechanism of the propeptide of the lipase. Conventional molecular dynamics (MD) and metadynamics simulations were used to explore the mechanism by which the propeptide affects the activity of the lipase, which was subsequently verified by mutation experiments. MD simulations indicated that the propeptide had an inhibitory effect on the lid movement of RCL and found a key region (Val5-Thr10) on the propeptide. Subsequently, site-directed mutations were created in this region. The mutations enhanced the lipase catalytic efficiency to 700% and showed the potential for the propeptide to shift the substrate specificity of RCL. The specificity and activity of RCL mutants also had similar trends to wild-type RCL toward triglycerides with varying chain lengths. The mutual corroboration of simulation and site-directed mutagenesis results revealed the vital role of the key propeptide region in the catalytic activity and substrate specificity of the lipase.