New insights into the molecular mechanism of methanol-induced inactivation of Thermomyces lanuginosus lipase: a molecular dynamics simulation study

Abstract

Methanol intolerance of lipase is a major limitation in lipase-catalysed methanolysis reactions. In this study, to understand the molecular mechanism of methanol-induced inactivation of lipases, we performed molecular dynamics (MD) simulations of Thermomyces lanuginosus lipase (TLL) in water and methanol and compared the observed structural and dynamic properties. The solvent accessibility analysis showed that in methanol, polar residues tended to be buried away from the solvent while non-polar residues tended to be more solvent-exposed in comparison to those in water. Moreover, we observed that in methanol, the van der Waals packing of the core residues in two hydrophobic regions of TLL became weak. Additionally, the catalytically relevant hydrogen bond between Asp²⁰¹ OD2 and His²⁵⁸ ND1 in the active site was broken when enzyme was solvated in methanol. This may affect the stability of the tetrahedral intermediates in the catalytic cycle of TLL. Furthermore, compared to in water, some enzyme surface residues displayed enhanced movement in methanol with higher Cα root-mean-square atomic positional fluctuation values. One of such methanol-affecting surface residues (Ile²⁴¹) was chosen for mutation, and MD simulation of the I241E mutant in methanol was conducted. The structural analysis of the mutant showed that replacing a non-polar surface residue with an acidic one at position 241 contributed to the stabilisation of enzyme structure in methanol. Ultimately, these results, while providing molecular-level insights into the destabilising effect of methanol on TLL, highlight the importance of surface residue redesign to improve the stability of lipases in methanol environments.

Keywords::

• Thermomyces lanuginosus lipase
• methanol intolerance
• molecular dynamics simulation
• rational design
• protein stability

Acknowledgements

We acknowledge the use of CPU times given by the EPSRC UK National Services for Computational Chemistry Software (NSCCS).

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

We acknowledge the financial support from the Doctoral School of Engineering and Science at Aalborg University and Otto Mønsteds Fond to Xiaoxue Tong for her visit to the University of Greenwich. This work was supported by Aalborg University, Faculty of Science and Technology and by the BioValue/SPIR grant DSF 0603-00522B from the Danish Council for strategic research and the Danish Council for technology and innovation.