Characterization and engineering of a two-enzyme system for plastics depolymerization.

Autor:	Knott BC; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Erickson E; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Allen MD; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom., Gado JE; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401.; Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506., Graham R; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom., Kearns FL; Department of Chemistry, University of South Florida, Tampa, FL 33620., Pardo I; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Topuzlu E; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401.; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717., Anderson JJ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Austin HP; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom., Dominick G; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Johnson CW; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Rorrer NA; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Szostkiewicz CJ; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Copié V; Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717., Payne CM; Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY 40506., Woodcock HL; Department of Chemistry, University of South Florida, Tampa, FL 33620., Donohoe BS; Biosciences Center, National Renewable Energy Laboratory, Golden, CO 80401., Beckham GT; Renewable Resources and Enabling Sciences Center, National Renewable Energy Laboratory, Golden, CO 80401; gregg.beckham@nrel.gov john.mcgeehan@port.ac.uk., McGeehan JE; Centre for Enzyme Innovation, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom; gregg.beckham@nrel.gov john.mcgeehan@port.ac.uk.
Jazyk:	angličtina
Zdroj:	Proceedings of the National Academy of Sciences of the United States of America [Proc Natl Acad Sci U S A] 2020 Oct 13; Vol. 117 (41), pp. 25476-25485. Date of Electronic Publication: 2020 Sep 28.
DOI:	10.1073/pnas.2006753117
Abstrakt:	Plastics pollution represents a global environmental crisis. In response, microbes are evolving the capacity to utilize synthetic polymers as carbon and energy sources. Recently, Ideonella sakaiensis was reported to secrete a two-enzyme system to deconstruct polyethylene terephthalate (PET) to its constituent monomers. Specifically, the I. sakaiensis PETase depolymerizes PET, liberating soluble products, including mono(2-hydroxyethyl) terephthalate (MHET), which is cleaved to terephthalic acid and ethylene glycol by MHETase. Here, we report a 1.6 Å resolution MHETase structure, illustrating that the MHETase core domain is similar to PETase, capped by a lid domain. Simulations of the catalytic itinerary predict that MHETase follows the canonical two-step serine hydrolase mechanism. Bioinformatics analysis suggests that MHETase evolved from ferulic acid esterases, and two homologous enzymes are shown to exhibit MHET turnover. Analysis of the two homologous enzymes and the MHETase S131G mutant demonstrates the importance of this residue for accommodation of MHET in the active site. We also demonstrate that the MHETase lid is crucial for hydrolysis of MHET and, furthermore, that MHETase does not turnover mono(2-hydroxyethyl)-furanoate or mono(2-hydroxyethyl)-isophthalate. A highly synergistic relationship between PETase and MHETase was observed for the conversion of amorphous PET film to monomers across all nonzero MHETase concentrations tested. Finally, we compare the performance of MHETase:PETase chimeric proteins of varying linker lengths, which all exhibit improved PET and MHET turnover relative to the free enzymes. Together, these results offer insights into the two-enzyme PET depolymerization system and will inform future efforts in the biological deconstruction and upcycling of mixed plastics. Competing Interests: Competing interest statement: A patent application was filed on this work. (Copyright © 2020 the Author(s). Published by PNAS.)
Databáze:	MEDLINE
Externí odkaz:	Zobrazit plný text záznamu