Graham R

References (6)

Title : Concentration-dependent inhibition of mesophilic PETases on poly(ethylene terephthalate) can be eliminated by enzyme engineering - Avilan_2023_ChemSusChem__e202202277
Author(s) : Avilan L , Lichtenstein BR , Koenig G , Zahn M , Allen MD , Oliveira L , Clark M , Bemmer V , Graham R , Austin HP , Dominick G , Johnson CW , Beckham GT , McGeehan J , Pickford AR
Ref : ChemSusChem , :e202202277 , 2023
Abstract : Enzyme-based depolymerization is a viable approach for recycling of poly(ethylene terephthalate) (PET). PETase from Ideonella sakaiensis (IsPETase) is capable of PET hydrolysis under mild conditions but suffers from concentration-dependent inhibition. Here, we report that this inhibition is dependent on incubation time, the solution conditions and PET surface area. Furthermore, this inhibition is evident in other mesophilic PET-degrading enzymes to varying degrees, independent of the level of PET depolymerization activity. The inhibition has no clear structural basis, but moderately thermostable IsPETase variants exhibit reduced inhibition, and the property is completely absent in the highly thermostable HotPETase, previously engineered by directed evolution, which our simulations suggest results from reduced flexibility around the active site. This work highlights a limitation in applying natural mesophilic hydrolases for PET hydrolysis, and reveals an unexpected positive outcome of engineering these enzymes for enhanced thermostability.
ESTHER : Avilan_2023_ChemSusChem__e202202277
PubMedSearch : Avilan_2023_ChemSusChem__e202202277
PubMedID: 36811288
Gene_locus related to this paper: psea4-u2z2l5 , 9gamm-a0a031mkr8 , 9burk-a0a1w6l438 , 9psed-a0a078mgg8 , pseol-e9kjl1 , 9burk-a0a0g3bi90 , 9burk-a0a1w6l588 , idesa-peth , acide-PBSA

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 : 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 : Comparative genomics and proteomics of Helicobacter mustelae, an ulcerogenic and carcinogenic gastric pathogen - O'Toole_2010_BMC.Genomics_11_164
Author(s) : O'Toole PW , Snelling WJ , Canchaya C , Forde BM , Hardie KR , Josenhans C , Graham R , McMullan G , Parkhill J , Belda E , Bentley SD
Ref : BMC Genomics , 11 :164 , 2010
Abstract : BACKGROUND: Helicobacter mustelae causes gastritis, ulcers and gastric cancer in ferrets and other mustelids. H. mustelae remains the only helicobacter other than H. pylori that causes gastric ulceration and cancer in its natural host. To improve understanding of H. mustelae pathogenesis, and the ulcerogenic and carcinogenic potential of helicobacters in general, we sequenced the H. mustelae genome, and identified 425 expressed proteins in the envelope and cytosolic proteome. RESULTS: The H. mustelae genome lacks orthologs of major H. pylori virulence factors including CagA, VacA, BabA, SabA and OipA. However, it encodes ten autotransporter surface proteins, seven of which were detected in the expressed proteome, and which, except for the Hsr protein, are of unknown function. There are 26 putative outer membrane proteins in H. mustelae, some of which are most similar to the Hof proteins of H. pylori. Although homologs of putative virulence determinants of H. pylori (NapA, plasminogen adhesin, collagenase) and Campylobacter jejuni (CiaB, Peb4a) are present in the H. mustelae genome, it also includes a distinct complement of virulence-related genes including a haemagglutinin/haemolysin protein, and a glycosyl transferase for producing blood group A/B on its lipopolysaccharide. The most highly expressed 264 proteins in the cytosolic proteome included many corresponding proteins from H. pylori, but the rank profile in H. mustelae was distinctive. Of 27 genes shown to be essential for H. pylori colonization of the gerbil, all but three had orthologs in H. mustelae, identifying a shared set of core proteins for gastric persistence. CONCLUSIONS: The determination of the genome sequence and expressed proteome of the ulcerogenic species H mustelae provides a comparative model for H. pylori to investigate bacterial gastric carcinogenesis in mammals, and to suggest ways whereby cag minus H. pylori strains might cause ulceration and cancer. The genome sequence was deposited in EMBL/GenBank/DDBJ under accession number FN555004.
ESTHER : O'Toole_2010_BMC.Genomics_11_164
PubMedSearch : O'Toole_2010_BMC.Genomics_11_164
PubMedID: 20219135
Gene_locus related to this paper: helm1-d3uha6 , helm1-d3uix4