Computational Insights into the Allosteric Modulation of a Phthalate-Degrading Hydrolase by Distal Mutations

Phthalate esters (PAEs) are a ubiquitous kind of environmental endocrine that disrupt chemicals, causing environmental and health issues. EstJ6 is an effective phthalate-degrading hydrolase, and its mutant with a combination of three non-conservative distal mutations has an improved activity against PAEs with unknown molecular mechanisms. Herein, we attempt to fill the significant gap between distal mutations and the activity of this enzyme using computational approaches. We found that mutations resulted in a redistribution of the enzyme's preexisting conformational states and dynamic changes of key functional regions, especially the lid over the active site. The outward motion of the lid upon the mutations made it easier for substrates or products to enter or exit. Additionally, a stronger substrate binding affinity and conformational rearrangements of catalytic reaction-associated residues in the mutant, accompanied by the strengthened communication within the protein, could synergistically contribute to the elevated catalytic efficiency. Finally, an attempt was made to improve the thermostability of EstJ6 upon introducing a distal disulfide bond between residues A23 and A29, and the simulation results were as expected. Together, our work explored the allosteric effects caused by distal mutations, which could provide insights into the rational design of esterases for industrial applications in the future.

Automated summary

In this study, computational approaches were used to investigate the molecular mechanisms behind the improved activity of a phthalate-degrading enzyme with three distal mutations. The mutations caused changes in the enzyme's conformational states and key functional regions, facilitating substrate binding and catalytic efficiency. Furthermore, the introduction of a distal disulfide bond improved the thermostability of the enzyme. Overall, this work provides insights into the rational design of esterases for industrial applications.