McGeehan JE

References (7)

Title : The reaction mechanism of the Ideonella sakaiensis PETase enzyme - Burgin_2024_Commun.Chem_7_65
Author(s) : Burgin T , Pollard BC , Knott BC , Mayes HB , Crowley MF , McGeehan JE , Beckham GT , Woodcock HL
Ref : Commun Chem , 7 :65 , 2024
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. To elucidate the multi-step PETase catalytic mechanism, we use transition path sampling and likelihood maximization to identify optimal reaction coordinates for the PETase enzyme. We predict that deacylation is likely rate-limiting, and the reaction coordinates for both steps include elements describing nucleophilic attack, ester bond cleavage, and the "moving-histidine" mechanism. We find that the flexibility of Trp185 promotes the reaction, providing an explanation for decreased activity observed in mutations that restrict Trp185 motion. Overall, this study uses unbiased computational approaches to reveal the detailed reaction mechanism necessary for further engineering of an important class of enzymes for plastics bioconversion.
ESTHER : Burgin_2024_Commun.Chem_7_65
PubMedSearch : Burgin_2024_Commun.Chem_7_65
PubMedID: 38538850
Gene_locus related to this paper: idesa-peth

Title : Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product Accumulation - Erickson_2022_ChemSusChem_15_e202102517
Author(s) : Erickson E , Shakespeare TJ , Bratti F , Buss BL , Graham R , Hawkins MA , Konig G , Michener WE , Miscall J , Ramirez KJ , Rorrer NA , Zahn M , Pickford AR , McGeehan JE , Beckham GT
Ref : ChemSusChem , 15 :e202102517 , 2022
Abstract : Invited for this month's cover is the BOTTLE Consortium, featuring Gregg Beckham's laboratory from NREL and John McGeehan's laboratory from the University of Portsmouth. The cover image shows the application of poly(ethylene terephthalate) (PET) hydrolase enzymes on post-consumer waste plastic, towards the development of an enzymatic PET recycling strategy. The Full Paper itself is available at 10.1002/cssc.202101932.
ESTHER : Erickson_2022_ChemSusChem_15_e202102517
PubMedSearch : Erickson_2022_ChemSusChem_15_e202102517
PubMedID: 34914860

Title : Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity - Erickson_2022_Nat.Commun_13_7850
Author(s) : Erickson E , Gado JE , Avilan L , Bratti F , Brizendine RK , Cox PA , Gill R , Graham R , Kim DJ , Konig G , Michener WE , Poudel S , Ramirez KJ , Shakespeare TJ , Zahn M , Boyd ES , Payne CM , Dubois JL , Pickford AR , Beckham GT , McGeehan JE
Ref : Nat Commun , 13 :7850 , 2022
Abstract : Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases. We successfully express, purify, and assay 51 enzymes from seven distinct phylogenetic groups; observing PET hydrolysis activity on amorphous PET film from 37 enzymes in reactions spanning pH from 4.5-9.0 and temperatures from 30-70 degreesC. We conduct PET hydrolysis time-course reactions with the best-performing enzymes, where we observe differences in substrate selectivity as function of PET morphology. We employed X-ray crystallography and AlphaFold to examine the enzyme architectures of all 74 candidates, revealing protein folds and accessory domains not previously associated with PET deconstruction. Overall, this study expands the number and diversity of thermotolerant scaffolds for enzymatic PET deconstruction.
ESTHER : Erickson_2022_Nat.Commun_13_7850
PubMedSearch : Erickson_2022_Nat.Commun_13_7850
PubMedID: 36543766
Gene_locus related to this paper: 9arch-PETcan211 , 9cren-PETcan204 , 9cyan-305pEE028 , 9bact-102Pee006 , 9chlr-7QJM202 , 9bact-a0a656d8b6 , 9actn-a0a1t4kk94 , 9burk-PET11 , 9bact-c3ryl0 , thecs-711Erick , 9actn-RII04304 , 9actn-h6wx58 , thecd-d1a9g5 , thecd-d1a2h1 , 9acto-d4q9n1 , 9acto-f7ix06 , 9gamm-a0a3l8bw54 , 9actn-a0a0n0my27 , 9burk-a0a1e4lw26 , 9actn-Alr407 , 9gamm-a0a3l8bdt3 , 9gamm-a0a2k9lit3 , 9bact-g9by57 , bacsu-pnbae , thefu-q6a0i4 , 9actn-a0a0n0ney5 , 9pseu-a0a1i6nu60 , thefu-q6a0i3 , 9actn-a0a147kjy8 , 9actn-e9upm2

Title : Comparative performance of PETase as a function of reaction conditions, substrate properties, and product accumulation - Erickson_2022_ChemSusChem_15_e202101932
Author(s) : Erickson E , Shakespeare TJ , Bratti F , Buss BL , Graham R , Hawkins MA , Konig G , Michener WE , Miscall J , Ramirez KJ , Rorrer NA , Zahn M , Pickford AR , McGeehan JE , Beckham G
Ref : ChemSusChem , 15 : , 2022
Abstract : There is keen interest to develop new technologies to recycle the plastic poly(ethylene terephthalate) (PET). To this end, the use of PET-hydrolyzing enzymes has shown promise for PET deconstruction to its monomers, terephthalate (TPA) and ethylene glycol (EG). Here, we compare the Ideonella sakaiensis PETase wild-type enzyme to a previously reported improved variant (W159H/S238F). We compare the thermostability of each enzyme and describe a 1.45 A resolution structure of the mutant, highlighting changes in the substrate binding cleft compared to the wild-type enzyme. Subsequently, the performance of the wild-type and variant enzyme was compared as a function of temperature, substrate morphology, and reaction mixture composition. These studies show that reaction temperature has the strongest influence on performance between the two enzymes. We also show that both enzymes achieve higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, we assess the impact of product accumulation on reaction progress for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displays different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both are sensitive to inhibition by EG. Overall, this study highlights the importance of reaction conditions, substrate selection, and product accumulation for catalytic performance of PET-hydrolyzing enzymes, which have implications for enzyme screening in the development of enzyme- based polyester recycling.
ESTHER : Erickson_2022_ChemSusChem_15_e202101932
PubMedSearch : Erickson_2022_ChemSusChem_15_e202101932
PubMedID: 34587366
Gene_locus related to this paper: idesa-peth

Title : A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis - Lusty_2022_RSC.Adv_12_8119
Author(s) : Lusty Beech J , Clare R , Kincannon WM , Erickson E , McGeehan JE , Beckham GT , Dubois JL
Ref : RSC Adv , 12 :8119 , 2022
Abstract : Esterase enzymes catalyze diverse hydrolysis reactions with important biological, commercial, and biotechnological applications. For the improvement of these biocatalysts, there is a need for widely accessible, inexpensive, and adaptable activity screening assays that identify enzymes with particular substrate specificities. Natural systems for biopolymer bioconversion, and likely those designed to mimic them, depend on cocktails of enzymes, each of which specifically targets the intact material as well as water-soluble subunits of varying size. In this work, we have adapted a UV/visible assay using pH-sensitive sulfonphthalein dyes for the real-time quantification of ester hydrolysis of bis-(2-hydroxyethyl) terephthalate (BHET), a subunit of polyethylene terephthalate (PET) plastic. We applied this method to a diverse set of known PET hydrolases and commercial esterases in a microplate format. The approach identified four PET hydrolases and one commercial esterase with high levels of specificity for BHET hydrolysis. Five additional PET hydrolases and three commercial esterases, including a thermophilic enzyme, effectively hydrolyzed both BHET and its monoester product MHET (mono-(2-hydroxyethyl) terephthalate). Specific activities were discernible within one hour and reactions reached an unequivocal endpoint well within 24 hours. The results from the UV/visible method correlated well with conventional HPLC analysis of the reaction products. We examined the suitability of the method toward variable pH, temperature, enzyme preparation method, mono- and multi-ester substrate type, and level of sensitivity versus stringency, finding the assay to be easily adaptable to diverse screening conditions and kinetic measurements. This method offers an accurate, easily accessible, and cost-effective route towards high-throughput library screening to support the discovery, directed evolution, and protein engineering of these critical biocatalysts.
ESTHER : Lusty_2022_RSC.Adv_12_8119
PubMedSearch : Lusty_2022_RSC.Adv_12_8119
PubMedID: 35424733

Title : Characterization and engineering of a two-enzyme system for plastics depolymerization - Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
Author(s) : Knott BC , Erickson E , Allen MD , Gado JE , Graham R , Kearns FL , Pardo I , Topuzlu E , Anderson JJ , Austin HP , Dominick G , Johnson CW , Rorrer NA , Szostkiewicz CJ , Copie V , Payne CM , Woodcock HL , Donohoe BS , Beckham GT , McGeehan JE
Ref : Proc Natl Acad Sci U S A , 117 :25476 , 2020
Abstract : 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 A 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.
ESTHER : Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
PubMedSearch : Knott_2020_Proc.Natl.Acad.Sci.U.S.A_117_25476
PubMedID: 32989159
Gene_locus related to this paper: idesa-mheth

Title : Characterization and engineering of a plastic-degrading aromatic polyesterase - Austin_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4350
Author(s) : Austin HP , Allen MD , Donohoe BS , Rorrer NA , Kearns FL , Silveira RL , Pollard BC , Dominick G , Duman R , El Omari K , Mykhaylyk V , Wagner A , Michener WE , Amore A , Skaf MS , Crowley MF , Thorne AW , Johnson CW , Woodcock HL , McGeehan JE , Beckham GT
Ref : Proc Natl Acad Sci U S A , 115 :E4350 , 2018
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 A resolution X-ray crystal structure of PETase, which reveals features common to both cutinases and lipases. PETase retains the ancestral alpha/beta-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.
ESTHER : Austin_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4350
PubMedSearch : Austin_2018_Proc.Natl.Acad.Sci.U.S.A_115_E4350
PubMedID: 29666242
Gene_locus related to this paper: idesa-peth