The effect of mutations in the lid region of Thermomyces lanuginosus lipase on interactions with triglyceride surfaces: A multi-scale simulation study

Abstract

Lipases naturally function at the interface formed between amphiphilic molecules and the aqueous environment. Thermomyces lanuginosus lipase (TLL) is a well-characterised lipase, known to exhibit interfacial activation during which a lid region covering the active site becomes displaced upon interaction with an interface. In this study, we investigate the effect the amino acid sequence of the lid region on interfacial binding and lid dynamics of TLL. Three TLL variants were investigated, a wild-type variant, a variant containing an esterase lid region (Esterase), and a Hybrid variant, containing both wild-type lid residues and esterase lid residues. Multiple coarse-grained molecular dynamics simulations revealed that the interfacial binding orientation of TLL was significantly affected by the nature of amino acids in the lid region, and atomistic simulations indicated effects on the structural dynamics of the lid itself. The atomistic simulations, as well as steered molecular dynamics simulations, also indicated that the Esterase lid region was less flexible than the wild-type lid region, whereas the Hybrid variant displayed superior lid flexibility and stability in the open conformation both at the interface, and in aqueous solution. Additional experiments performed to investigate the activity and binding behaviour of the lipase variants indicated a slightly higher specific activity for the Hybrid variant compared to the wild-type variant, correlating the observations of increased lid flexibility. Together, these results are in line with previous experimental studies, highlighting the importance of the nature of the amino acid residues within the functional lid region of lipases, particularly regarding interfacial binding orientation, activation, and structural stability.

Direct observation of Thermomyces lanuginosus lipase diffusional states by Single Particle Tracking and their remodeling by mutations and inhibition

“…We next quantified how TLL docking and diffusional properties depend on lid mutations that we have recently showed to affect lid dynamics and function (see Fig. 2A,B) 10,11,13 . L2 contains ferulic acid esterase (FAEA) lid, while L3 had a hybrid lid composition of both FAEA and TLL character.…”

“…This mutant has been shown to exhibit much lower activity than native 10,13 , albeit having a more open lid and therefore displaying an interfacially independent activity 10,11 . Additionally, recent MD simulation studies indicate L2 to have the least flexible lid conformation 13 when compared to native. L3 variant on the other hand is rationally designed to have a hybrid lid, with structural origins from both native TLL and FAEA 12 .…”

“…L3 variant on the other hand is rationally designed to have a hybrid lid, with structural origins from both native TLL and FAEA 12 . Interestingly, this mutant was later found to have a more dynamic lid than native TLL, activate more favorably under conditions mimicking interfacial activation (low polarity solvent) 11 and, possibly as a corollary of these, attain a slightly higher activity under some conditions 13 .…”

Enzymatic transesterification of urethane-bond containing ester

“…For the other tested lipases from Candida antarctica [49][50][51][52][53], Thermomyces lanuginosa [54][55][56][57], Rhizomucor Fig. 7 Biocatalyst effect: educt amounts and product yields after transesterification catalyzed by different immobilized lipases.…”

Catalytic Activation of Esterases by PEGylation for Polyester Synthesis

“…Recently, MD studies and other modelling techniques have been used to help understand the mechanisms of interfacial activation in lipase TL, CALB and other lipases. [23] However, these studies are in general performed in water medium, or in a water-lipid/water-oil interface. We show herein an innovative way to demonstrate the contribution of enzyme PEGylation on the final enzyme arrangement.…”