The reaction mechanism of the Ideonella sakaiensis PETase enzyme

Tucker Burgin, Benjamin C. Pollard, Brandon C. Knott, Heather, B. Mayes, Michael F. Crowley, John McGeehan, Gregg T. Beckham*, H. Lee Woodcock*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

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Abstract

Polyethylene terephthalate (PET), the most abundantly produced polyester plastic, can be depolymerized by the Ideonella sakaiensis PETase enzyme. Based on multiple PETase crystal structures, the reaction has been proposed to proceed via a two-step serine hydrolase mechanism mediated by a serine-histidine-aspartate catalytic triad. However, the details and rate-limiting step remained heretofore unknown, with speculation regarding the role of ancillary residues along the catalytic itinerary. To elucidate the multi-step PETase catalytic mechanism, we use path sampling and likelihood maximization to identify optimal reaction coordinates. We predict that acylation is rate-limiting and the reaction coordinates for both steps include elements describing nucleophilic attack, ester cleavage, and the "moving-histidine" mechanism. We find the flexibility of Trp185 is critical to the reaction, providing an explanation for decreased activity observed in mutations restricting Trp185 motion. Overall, this study reveals the detailed reaction mechanism necessary towards further engineering an important class of enzymes for plastics bioconversion.
Original languageEnglish
Article number65
Number of pages14
JournalCommunications Chemistry
Volume7
DOIs
Publication statusPublished - 27 Mar 2024

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