Characterization of a cytochrome P450 that catalyzes the O-demethylation of lignin-derived benzoates.

Cytochromes P450 (P450s) are a superfamily of heme-containing enzymes possessing a broad range of monooxygenase activities. One such activity is O-demethylation, an essential and rate-determining step in emerging strategies to valorize lignin that employ carbon-carbon bond cleavage. We recently identified PbdA, a P450 from Rhodococcus jostii RHA1, and PbdB, its cognate reductase, which catalyze the O-demethylation of para-methoxylated benzoates (p-MBAs) to initiate growth of RHA1 on these compounds. PbdA had the highest affinity (Kd = 3.8 ± 0.6 µM) and apparent specificity (kcat/KM = 20,000 ± 3000 M-1 s-1) for p-MBA. The enzyme also O-demethylated two related lignin-derived aromatic compounds with remarkable efficiency: veratrate and isovanillate. PbdA also catalyzed the hydroxylation and dehydrogenation of p-ethylbenzoate even though RHA1 did not grow on this compound. Atomic-resolution structures of PbdA in complex with p-MBA, p-ethylbenzoate, and veratrate revealed a cluster of three residues that form hydrogen bonds with the substrates' carboxylate: Ser87, Ser237, and Arg84. Substitution of these residues resulted in lower affinity and O-demethylation activity on p-MBA as well as increased affinity for the acetyl analog, p-methoxyacetophenone. The S87A and S237A variants of PbdA also catalyzed the O-demethylation of an aldehyde analog of p-MBA, p-methoxy-benzaldehyde, while the R84M variant did not, despite binding this compound with high affinity. These results suggest that Ser87, Ser237, and Arg84 are not only important determinants of specificity but also help to orientate that substrate correctly in the active site. This study facilitates the design of biocatalysts for lignin valorization.

The reaction mechanism of the Ideonella sakaiensis PETase enzyme.

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.

Initiation of fatty acid biosynthesis in Pseudomonas putida KT2440.

Deciphering the mechanisms of bacterial fatty acid biosynthesis is crucial for both the engineering of bacterial hosts to produce fatty acid-derived molecules and the development of new antibiotics. However, gaps in our understanding of the initiation of fatty acid biosynthesis remain. Here, we demonstrate that the industrially relevant microbe Pseudomonas putida KT2440 contains three distinct pathways to initiate fatty acid biosynthesis. The first two routes employ conventional ß-ketoacyl-ACP synthase III enzymes, FabH1 and FabH2, that accept short- and medium-chain-length acyl-CoAs, respectively. The third route utilizes a malonyl-ACP decarboxylase enzyme, MadB. A combination of exhaustive in vivo alanine-scanning mutagenesis, in vitro biochemical characterization, X-ray crystallography, and computational modeling elucidate the presumptive mechanism of malonyl-ACP decarboxylation via MadB. Given that functional homologs of MadB are widespread throughout domain Bacteria, this ubiquitous alternative fatty acid initiation pathway provides new opportunities to target a range of biotechnology and biomedical applications.

Concentration-Dependent Inhibition of Mesophilic PETases on Poly(ethylene terephthalate) Can Be Eliminated by Enzyme Engineering.

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. In this study, this inhibition is found to be 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 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.

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro-1,2-diguaiacylpropane-1,3-diol (erythro-DGPD), a ring-opened lignin-derived ß-1 dimer, was reported in Novosphingobium aromaticivorans. The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro-DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C1 reaction product, and we further demonstrated that both enantiomers of erythro-DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

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 °C. 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.

A flexible kinetic assay efficiently sorts prospective biocatalysts for PET plastic subunit hydrolysis.

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.

Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism.

SignificanceMore than 400 million tons of plastic waste is produced each year, the overwhelming majority of which ends up in landfills. Bioconversion strategies aimed at plastics have emerged as important components of enabling a circular economy for synthetic plastics, especially those that exhibit chemically similar linkages to those found in nature, such as polyesters. The enzyme system described in this work is essential for mineralization of the xenobiotic components of poly(ethylene terephthalate) (PET) in the biosphere. Our description of its structure and substrate preferences lays the groundwork for in vivo or ex vivo engineering of this system for PET upcycling.

Characterization of a phylogenetically distinct extradiol dioxygenase involved in the bacterial catabolism of lignin-derived aromatic compounds.

The actinobacterium Rhodococcus jostii RHA1 grows on a remarkable variety of aromatic compounds and has been studied for applications ranging from the degradation of polychlorinated biphenyls to the valorization of lignin, an underutilized component of biomass. In RHA1, the catabolism of two classes of lignin-derived compounds, alkylphenols and alkylguaiacols, involves a phylogenetically distinct extradiol dioxygenase, AphC, previously misannotated as BphC, an enzyme involved in biphenyl catabolism. To better understand the role of AphC in RHA1 catabolism, we first showed that purified AphC had highest apparent specificity for 4-propylcatechol (kcat/KM ∼106 M-1 s-1), and its apparent specificity for 4-alkylated substrates followed the trend for alkylguaiacols: propyl > ethyl > methyl > phenyl > unsubstituted. We also show AphC only poorly cleaved 3-phenylcatechol, the preferred substrate of BphC. Moreover, AphC and BphC cleaved 3-phenylcatechol and 4-phenylcatechol with different regiospecificities, likely due to the substrates' binding mode. A crystallographic structure of the AphC·4-ethylcatechol binary complex to 1.59 Å resolution revealed that the catechol is bound to the active site iron in a bidentate manner and that the substrate's alkyl side chain is accommodated by a hydrophobic pocket. Finally, we show RHA1 grows on a mixture of 4-ethylguaiacol and guaiacol, simultaneously catabolizing these substrates through meta-cleavage and ortho-cleavage pathways, respectively, suggesting that the specificity of AphC helps to prevent the routing of catechol through the Aph pathway. Overall, this study contributes to our understanding of the bacterial catabolism of aromatic compounds derived from lignin, and the determinants of specificity in extradiol dioxygenases.

Debottlenecking 4-hydroxybenzoate hydroxylation in Pseudomonas putida KT2440 improves muconate productivity from p-coumarate.

The transformation of 4-hydroxybenzoate (4-HBA) to protocatechuate (PCA) is catalyzed by flavoprotein oxygenases known as para-hydroxybenzoate-3-hydroxylases (PHBHs). In Pseudomonas putida KT2440 (P. putida) strains engineered to convert lignin-related aromatic compounds to muconic acid (MA), PHBH activity is rate-limiting, as indicated by the accumulation of 4-HBA, which ultimately limits MA productivity. Here, we hypothesized that replacement of PobA, the native P. putida PHBH, with PraI, a PHBH from Paenibacillus sp. JJ-1b with a broader nicotinamide cofactor preference, could alleviate this bottleneck. Biochemical assays confirmed the strict preference of NADPH for PobA, while PraI can utilize either NADH or NADPH. Kinetic assays demonstrated that both PobA and PraI can utilize NADPH with comparable catalytic efficiency and that PraI also efficiently utilizes NADH at roughly half the catalytic efficiency. The X-ray crystal structure of PraI was solved and revealed absolute conservation of the active site architecture to other PHBH structures despite their differing cofactor preferences. To understand the effect in vivo, we compared three P. putida strains engineered to produce MA from p-coumarate (pCA), showing that expression of praI leads to lower 4-HBA accumulation and decreased NADP+/NADPH ratios relative to strains harboring pobA, indicative of a relieved 4-HBA bottleneck due to increased NADPH availability. In bioreactor cultivations, a strain exclusively expressing praI achieved a titer of 40 g/L MA at 100% molar yield and a productivity of 0.5 g/L/h. Overall, this study demonstrates the benefit of sampling readily available natural enzyme diversity for debottlenecking metabolic flux in an engineered strain for microbial conversion of lignin-derived compounds to value-added products.

Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product Accumulation.

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.

Cytochromes P450 in the biocatalytic valorization of lignin.

The valorization of lignin is critical to establishing sustainable biorefineries as we transition away from petroleum-derived feedstocks. Advances in lignin fractionation and depolymerization are yielding new opportunities for the biocatalytic upgrading of lignin-derived aromatic compounds (LDACs) using microbial cell factories. Given their roles in lignin metabolism and their catalytic versatility, cytochromes P450 are attractive enzymes in engineering such biocatalysts. Here we highlight P450s that catalyze aromatic O-demethylation, a rate-limiting step in the conversion of LDACs to valuable chemicals, including efforts to engineer the specificity of these enzymes and to use them in developing biocatalysts. We also discuss broader opportunities at the intersection of biochemistry, structure-guided enzyme engineering, and metabolic engineering for application of P450s in the emerging area of microbial lignin valorization.

Comparative Performance of PETase as a Function of Reaction Conditions, Substrate Properties, and Product Accumulation.

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, the Ideonella sakaiensis PETase wild-type enzyme was compared to a previously reported improved variant (W159H/S238F). The thermostability of each enzyme was compared and a 1.45 Šresolution structure of the mutant was described, 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 showed that reaction temperature had the strongest influence on performance between the two enzymes. It was also shown that both enzymes achieved higher levels of PET conversion for substrates with moderate crystallinity relative to amorphous substrates. Finally, the impact of product accumulation on reaction progress was assessed for the hydrolysis of both PET and bis(2-hydroxyethyl) terephthalate (BHET). Each enzyme displayed different inhibition profiles to mono(2-hydroxyethyl) terephthalate (MHET) and TPA, while both were 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.

Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.

Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

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

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.

Integrated Approach Reveals Role of Mitochondrial Germ-Line Mutation F18L in Respiratory Chain, Oxidative Alterations, Drug Sensitivity, and Patient Prognosis in Glioblastoma.

Glioblastoma is the most common and malignant primary brain tumour in adults, with a dismal prognosis. This is partly due to considerable inter- and intra-tumour heterogeneity. Changes in the cellular energy-producing mitochondrial respiratory chain complex (MRC) activities are a hallmark of glioblastoma relative to the normal brain, and associate with differential survival outcomes. Targeting MRC complexes with drugs can also facilitate anti-glioblastoma activity. Whether mutations in the mitochondrial DNA (mtDNA) that encode several components of the MRC contribute to these phenomena remains underexplored. We identified a germ-line mtDNA mutation (m. 14798T > C), enriched in glioblastoma relative to healthy controls, that causes an amino acid substitution F18L within the core mtDNA-encoded cytochrome b subunit of MRC complex III. F18L is predicted to alter corresponding complex III activity, and sensitivity to complex III-targeting drugs. This could in turn alter reactive oxygen species (ROS) production, cell behaviour and, consequently, patient outcomes. Here we show that, despite a heterogeneous mitochondrial background in adult glioblastoma patient biopsy-derived cell cultures, the F18L substitution associates with alterations in individual MRC complex activities, in particular a 75% increase in MRC complex II_III activity, and a 34% reduction in CoQ10, the natural substrate for MRC complex III, levels. Downstream characterisation of an F18L-carrier revealed an 87% increase in intra-cellular ROS, an altered cellular distribution of mitochondrial-specific ROS, and a 64% increased sensitivity to clomipramine, a repurposed MRC complex III-targeting drug. In patients, F18L-carriers that received the current standard of care treatment had a poorer prognosis than non-carriers (373 days vs. 415 days, respectively). Single germ-line mitochondrial mutations could predispose individuals to differential prognoses, and sensitivity to mitochondrial targeted drugs. Thus, F18L, which is present in blood could serve as a useful non-invasive biomarker for the stratification of patients into prognostically relevant groups, one of which requires a lower dose of clomipramine to achieve clinical effect, thus minimising side-effects.

Enabling microbial syringol conversion through structure-guided protein engineering.

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is O-aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol O-demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

The Molecular Basis for Apolipoprotein E4 as the Major Risk Factor for Late-Onset Alzheimer's Disease.

Apolipoprotein E4 (ApoE4) is one of three (E2, E3 and E4) human isoforms of an α-helical, 299-amino-acid protein. Homozygosity for the ε4 allele is the major genetic risk factor for developing late-onset Alzheimer's disease (AD). ApoE2, ApoE3 and ApoE4 differ at amino acid positions 112 and 158, and these sequence variations may confer conformational differences that underlie their participation in the risk of developing AD. Here, we compared the shape, oligomerization state, conformation and stability of ApoE isoforms using a range of complementary biophysical methods including small-angle x-ray scattering, analytical ultracentrifugation, circular dichroism, x-ray fiber diffraction and transmission electron microscopy We provide an in-depth and definitive study demonstrating that all three proteins are similar in stability and conformation. However, we show that ApoE4 has a propensity to polymerize to form wavy filaments, which do not share the characteristics of cross-ß amyloid fibrils. Moreover, we provide evidence for the inhibition of ApoE4 fibril formation by ApoE3. This study shows that recombinant ApoE isoforms show no significant differences at the structural or conformational level. However, self-assembly of the ApoE4 isoform may play a role in pathogenesis, and these results open opportunities for uncovering new triggers for AD onset.

A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion.

Microbial aromatic catabolism offers a promising approach to convert lignin, a vast source of renewable carbon, into useful products. Aryl-O-demethylation is an essential biochemical reaction to ultimately catabolize coniferyl and sinapyl lignin-derived aromatic compounds, and is often a key bottleneck for both native and engineered bioconversion pathways. Here, we report the comprehensive characterization of a promiscuous P450 aryl-O-demethylase, consisting of a cytochrome P450 protein from the family CYP255A (GcoA) and a three-domain reductase (GcoB) that together represent a new two-component P450 class. Though originally described as converting guaiacol to catechol, we show that this system efficiently demethylates both guaiacol and an unexpectedly wide variety of lignin-relevant monomers. Structural, biochemical, and computational studies of this novel two-component system elucidate the mechanism of its broad substrate specificity, presenting it as a new tool for a critical step in biological lignin conversion.

Characterization and engineering of a plastic-degrading aromatic polyesterase.

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/ß-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.