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Characterization and engineering of a plastic-degrading aromatic polyesterase

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      Significance

      Synthetic polymers are ubiquitous in the modern world but pose a global environmental problem. While plastics such as poly(ethylene terephthalate) (PET) are highly versatile, their resistance to natural degradation presents a serious, growing risk to fauna and flora, particularly in marine environments. Here, we have characterized the 3D structure of a newly discovered enzyme that can digest highly crystalline PET, the primary material used in the manufacture of single-use plastic beverage bottles, in some clothing, and in carpets. We engineer this enzyme for improved PET degradation capacity and further demonstrate that it can also degrade an important PET replacement, polyethylene-2,5-furandicarboxylate, providing new opportunities for biobased plastics recycling.

      Abstract

      Poly(ethylene terephthalate) (PET) is one of the most abundantly produced synthetic polymers and is accumulating in the environment at a staggering rate as discarded packaging and textiles. The properties that make PET so useful also endow it with an alarming resistance to biodegradation, likely lasting centuries in the environment. Our collective reliance on PET and other plastics means that this buildup will continue unless solutions are found. Recently, a newly discovered bacterium, Ideonella sakaiensis 201-F6, was shown to exhibit the rare ability to grow on PET as a major carbon and energy source. Central to its PET biodegradation capability is a secreted PETase (PET-digesting enzyme). Here, we present a 0.92 Å resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral α/β-hydrolase fold but exhibits a more open active-site cleft than homologous cutinases. By narrowing the binding cleft via mutation of two active-site residues to conserved amino acids in cutinases, we surprisingly observe improved PET degradation, suggesting that PETase is not fully optimized for crystalline PET degradation, despite presumably evolving in a PET-rich environment. Additionally, we show that PETase degrades another semiaromatic polyester, polyethylene-2,5-furandicarboxylate (PEF), which is an emerging, bioderived PET replacement with improved barrier properties. In contrast, PETase does not degrade aliphatic polyesters, suggesting that it is generally an aromatic polyesterase. These findings suggest that additional protein engineering to increase PETase performance is realistic and highlight the need for further developments of structure/activity relationships for biodegradation of synthetic polyesters.

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      Most cited references 70

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      Protein and ligand preparation: parameters, protocols, and influence on virtual screening enrichments.

      Structure-based virtual screening plays an important role in drug discovery and complements other screening approaches. In general, protein crystal structures are prepared prior to docking in order to add hydrogen atoms, optimize hydrogen bonds, remove atomic clashes, and perform other operations that are not part of the x-ray crystal structure refinement process. In addition, ligands must be prepared to create 3-dimensional geometries, assign proper bond orders, and generate accessible tautomer and ionization states prior to virtual screening. While the prerequisite for proper system preparation is generally accepted in the field, an extensive study of the preparation steps and their effect on virtual screening enrichments has not been performed. In this work, we systematically explore each of the steps involved in preparing a system for virtual screening. We first explore a large number of parameters using the Glide validation set of 36 crystal structures and 1,000 decoys. We then apply a subset of protocols to the DUD database. We show that database enrichment is improved with proper preparation and that neglecting certain steps of the preparation process produces a systematic degradation in enrichments, which can be large for some targets. We provide examples illustrating the structural changes introduced by the preparation that impact database enrichment. While the work presented here was performed with the Protein Preparation Wizard and Glide, the insights and guidance are expected to be generalizable to structure-based virtual screening with other docking methods.
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        The alpha/beta hydrolase fold.

         M. Harel,  F Frolow,  I Silman (1992)
        We have identified a new protein fold--the alpha/beta hydrolase fold--that is common to several hydrolytic enzymes of widely differing phylogenetic origin and catalytic function. The core of each enzyme is similar: an alpha/beta sheet, not barrel, of eight beta-sheets connected by alpha-helices. These enzymes have diverged from a common ancestor so as to preserve the arrangement of the catalytic residues, not the binding site. They all have a catalytic triad, the elements of which are borne on loops which are the best-conserved structural features in the fold. Only the histidine in the nucleophile-histidine-acid catalytic triad is completely conserved, with the nucleophile and acid loops accommodating more than one type of amino acid. The unique topological and sequence arrangement of the triad residues produces a catalytic triad which is, in a sense, a mirror-image of the serine protease catalytic triad. There are now four groups of enzymes which contain catalytic triads and which are related by convergent evolution towards a stable, useful active site: the eukaryotic serine proteases, the cysteine proteases, subtilisins and the alpha/beta hydrolase fold enzymes.
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          Marine pollution. Plastic waste inputs from land into the ocean.

          Plastic debris in the marine environment is widely documented, but the quantity of plastic entering the ocean from waste generated on land is unknown. By linking worldwide data on solid waste, population density, and economic status, we estimated the mass of land-based plastic waste entering the ocean. We calculate that 275 million metric tons (MT) of plastic waste was generated in 192 coastal countries in 2010, with 4.8 to 12.7 million MT entering the ocean. Population size and the quality of waste management systems largely determine which countries contribute the greatest mass of uncaptured waste available to become plastic marine debris. Without waste management infrastructure improvements, the cumulative quantity of plastic waste available to enter the ocean from land is predicted to increase by an order of magnitude by 2025.
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            Author and article information

            Affiliations
            aMolecular Biophysics Laboratories, School of Biological Sciences, Institute of Biological and Biomedical Sciences, University of Portsmouth, Portsmouth PO1 2DY, United Kingdom;
            bBiosciences Center, National Renewable Energy Laboratory, Golden, CO 80401;
            cNational Bioenergy Center, National Renewable Energy Laboratory, Golden, CO 80401;
            dDepartment of Chemistry, University of South Florida, Tampa, FL 33620-5250;
            eInstitute of Chemistry, University of Campinas, Campinas, 13083-970 Sao Paulo, Brazil;
            fDiamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, United Kingdom
            Author notes
            2To whom correspondence may be addressed. Email: hlw@123456mail.usf.edu, john.mcgeehan@123456port.ac.uk, or gregg.beckham@123456nrel.gov.

            Edited by Alexis T. Bell, University of California, Berkeley, CA, and approved March 28, 2018 (received for review October 29, 2017)

            Author contributions: J.E.M. and G.T.B. designed research; H.P.A., M.D.A., B.S.D., N.A.R., F.L.K., R.L.S., B.C.P., G.D., R.D., K.E.O., V.M., A.W., W.E.M., A.A., A.W.T., C.W.J., and H.L.W. performed research; H.P.A., M.D.A., B.S.D., N.A.R., F.L.K., R.L.S., B.C.P., G.D., R.D., K.E.O., V.M., A.W., W.E.M., A.A., M.S.S., M.F.C., A.W.T., C.W.J., H.L.W., J.E.M., and G.T.B. analyzed data; and J.E.M. and G.T.B. wrote the paper with contributions from all authors.

            1H.P.A., M.D.A., B.S.D., N.A.R., and F.L.K. contributed equally to this work.

            Journal
            Proc Natl Acad Sci U S A
            Proc. Natl. Acad. Sci. U.S.A
            pnas
            pnas
            PNAS
            Proceedings of the National Academy of Sciences of the United States of America
            National Academy of Sciences
            0027-8424
            1091-6490
            8 May 2018
            17 April 2018
            17 April 2018
            : 115
            : 19
            : E4350-E4357
            29666242
            5948967
            201718804
            10.1073/pnas.1718804115
            Copyright © 2018 the Author(s). Published by PNAS.

            This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

            Counts
            Pages: 8
            Product
            Funding
            Funded by: NREL
            Award ID: LDRD
            Funded by: BBSRC
            Award ID: BB/P011918/1
            Funded by: Sao Paulo Research Foundation
            Award ID: #2016/22956-7
            Funded by: US Department of Energy
            Award ID: DE-SC0011297TDD
            Categories
            PNAS Plus
            Biological Sciences
            Biochemistry
            PNAS Plus
            Keywords:

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