Woodcock HL

References (3)

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 : 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