Engineering of glycoside hydrolase family 7 cellobiohydrolases directed by natural diversity screening.

Protein engineering and screening of processive fungal cellobiohydrolases (CBHs) remain challenging due to limited expression hosts, synergy-dependency, and recalcitrant substrates. In particular, glycoside hydrolase family 7 (GH7) CBHs are critically important for the bioeconomy and typically difficult to engineer. Here, we target the discovery of highly active natural GH7 CBHs and engineering of variants with improved activity. Using experimentally assayed activities of genome mined CBHs, we applied sequence and structural alignments to top performers to identify key point mutations linked to improved activity. From ∼1500 known GH7 sequences, an evolutionarily diverse subset of 57 GH7 CBH genes was expressed in Trichoderma reesei and screened using a multiplexed activity screening assay. Ten catalytically enhanced natural variants were identified, produced, purified, and tested for efficacy using industrially relevant conditions and substrates. Three key amino acids in CBHs with performance comparable or superior to Penicillium funiculosum Cel7A were identified and combinatorially engineered into P. funiculosum cel7a, expressed in T. reesei, and assayed on lignocellulosic biomass. The top performer generated using this combined approach of natural diversity genome mining, experimental assays, and computational modeling produced a 41% increase in conversion extent over native P. funiculosum Cel7A, a 55% increase over the current industrial standard T. reesei Cel7A, and 10% improvement over Aspergillus oryzae Cel7C, the best natural GH7 CBH previously identified in our laboratory.

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.

The dissociation mechanism of processive cellulases.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing. We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

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 hydrolysis mechanism of a GH45 cellulase and its potential relation to lytic transglycosylase and expansin function.

Family 45 glycoside hydrolases (GH45) are endoglucanases that are integral to cellulolytic secretomes, and their ability to break down cellulose has been successfully exploited in textile and detergent industries. In addition to their industrial relevance, understanding the molecular mechanism of GH45-catalyzed hydrolysis is of fundamental importance because of their structural similarity to cell wall-modifying enzymes such as bacterial lytic transglycosylases (LTs) and expansins present in bacteria, plants, and fungi. Our understanding of the catalytic itinerary of GH45s has been incomplete because a crystal structure with substrate spanning the -1 to +1 subsites is currently lacking. Here we constructed and validated a putative Michaelis complex in silico and used it to elucidate the hydrolytic mechanism in a GH45, Cel45A from the fungus Humicola insolens, via unbiased simulation approaches. These molecular simulations revealed that the solvent-exposed active-site architecture results in lack of coordination for the hydroxymethyl group of the substrate at the -1 subsite. This lack of coordination imparted mobility to the hydroxymethyl group and enabled a crucial hydrogen bond with the catalytic acid during and after the reaction. This suggests the possibility of a nonhydrolytic reaction mechanism when the catalytic base aspartic acid is missing, as is the case in some LTs (murein transglycosylase A) and expansins. We calculated reaction free energies and demonstrate the thermodynamic feasibility of the hydrolytic and nonhydrolytic reaction mechanisms. Our results provide molecular insights into the hydrolysis mechanism in HiCel45A, with possible implications for elucidating the elusive catalytic mechanism in LTs and expansins.

Distinct roles of N- and O-glycans in cellulase activity and stability.

In nature, many microbes secrete mixtures of glycoside hydrolases, oxidoreductases, and accessory enzymes to deconstruct polysaccharides and lignin in plants. These enzymes are often decorated with N- and O-glycosylation, the roles of which have been broadly attributed to protection from proteolysis, as the extracellular milieu is an aggressive environment. Glycosylation has been shown to sometimes affect activity, but these effects are not fully understood. Here, we examine N- and O-glycosylation on a model, multimodular glycoside hydrolase family 7 cellobiohydrolase (Cel7A), which exhibits an O-glycosylated carbohydrate-binding module (CBM) and an O-glycosylated linker connected to an N- and O-glycosylated catalytic domain (CD)-a domain architecture common to many biomass-degrading enzymes. We report consensus maps for Cel7A glycosylation that include glycan sites and motifs. Additionally, we examine the roles of glycans on activity, substrate binding, and thermal and proteolytic stability. N-glycan knockouts on the CD demonstrate that N-glycosylation has little impact on cellulose conversion or binding, but does have major stability impacts. O-glycans on the CBM have little impact on binding, proteolysis, or activity in the whole-enzyme context. However, linker O-glycans greatly impact cellulose conversion via their contribution to proteolysis resistance. Molecular simulations predict an additional role for linker O-glycans, namely that they are responsible for maintaining separation between ordered domains when Cel7A is engaged on cellulose, as models predict α-helix formation and decreased cellulose interaction for the nonglycosylated linker. Overall, this study reveals key roles for N- and O-glycosylation that are likely broadly applicable to other plant cell-wall-degrading enzymes.

Biochemical and Structural Characterizations of Two Dictyostelium Cellobiohydrolases from the Amoebozoa Kingdom Reveal a High Level of Conservation between Distant Phylogenetic Trees of Life.

UNLABELLED: Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) are enzymes commonly employed in plant cell wall degradation across eukaryotic kingdoms of life, as they provide significant hydrolytic potential in cellulose turnover. To date, many fungal GH7 CBHs have been examined, yet many questions regarding structure-activity relationships in these important natural and commercial enzymes remain. Here, we present the crystal structures and a biochemical analysis of two GH7 CBHs from social amoeba: Dictyostelium discoideum Cel7A (DdiCel7A) and Dictyostelium purpureum Cel7A (DpuCel7A). DdiCel7A and DpuCel7A natively consist of a catalytic domain and do not exhibit a carbohydrate-binding module (CBM). The structures of DdiCel7A and DpuCel7A, resolved to 2.1 Å and 2.7 Å, respectively, are homologous to those of other GH7 CBHs with an enclosed active-site tunnel. Two primary differences between the Dictyostelium CBHs and the archetypal model GH7 CBH, Trichoderma reesei Cel7A (TreCel7A), occur near the hydrolytic active site and the product-binding sites. To compare the activities of these enzymes with the activity of TreCel7A, the family 1 TreCel7A CBM and linker were added to the C terminus of each of the Dictyostelium enzymes, creating DdiCel7ACBM and DpuCel7ACBM, which were recombinantly expressed in T. reesei DdiCel7ACBM and DpuCel7ACBM hydrolyzed Avicel, pretreated corn stover, and phosphoric acid-swollen cellulose as efficiently as TreCel7A when hydrolysis was compared at their temperature optima. The Ki of cellobiose was significantly higher for DdiCel7ACBM and DpuCel7ACBM than for TreCel7A: 205, 130, and 29 µM, respectively. Taken together, the present study highlights the remarkable degree of conservation of the activity of these key natural and industrial enzymes across quite distant phylogenetic trees of life. IMPORTANCE: GH7 CBHs are among the most important cellulolytic enzymes both in nature and for emerging industrial applications for cellulose breakdown. Understanding the diversity of these key industrial enzymes is critical to engineering them for higher levels of activity and greater stability. The present work demonstrates that two GH7 CBHs from social amoeba are surprisingly quite similar in structure and activity to the canonical GH7 CBH from the model biomass-degrading fungus T. reesei when tested under equivalent conditions (with added CBM-linker domains) on an industrially relevant substrate.

Nucleation rate analysis of methane hydrate from molecular dynamics simulations.

Clathrate hydrates are solid crystalline structures most commonly formed from solutions that have nucleated to form a mixed solid composed of water and gas. Understanding the mechanism of clathrate hydrate nucleation is essential to grasp the fundamental chemistry of these complex structures and their applications. Molecular dynamics (MD) simulation is an ideal method to study nucleation at the molecular level because the size of the critical nucleus and formation rate occur on the nano scale. Various analysis methods for nucleation have been developed through MD to analyze nucleation. In particular, the mean first-passage time (MFPT) and survival probability (SP) methods have proven to be effective in procuring the nucleation rate and critical nucleus size for monatomic systems. This study assesses the MFPT and SP methods, previously used for monatomic systems, when applied to analyzing clathrate hydrate nucleation. Because clathrate hydrate nucleation is relatively difficult to observe in MD simulations (due to its high free energy barrier), these methods have yet to be applied to clathrate hydrate systems. In this study, we have analyzed the nucleation rate and critical nucleus size of methane hydrate using MFPT and SP methods from data generated by MD simulations at 255 K and 50 MPa. MFPT was modified for clathrate hydrate from the original version by adding the maximum likelihood estimate and growth effect term. The nucleation rates calculated by MFPT and SP methods are within 5%, and the critical nucleus size estimated by the MFPT method was 50% higher, than values obtained through other more rigorous but computationally expensive estimates. These methods can also be extended to the analysis of other clathrate hydrates.

Carbohydrate-protein interactions that drive processive polysaccharide translocation in enzymes revealed from a computational study of cellobiohydrolase processivity.

Translocation of carbohydrate polymers through protein tunnels and clefts is a ubiquitous biochemical phenomenon in proteins such as polysaccharide synthases, glycoside hydrolases, and carbohydrate-binding modules. Although static snapshots of carbohydrate polymer binding in proteins have long been studied via crystallography and spectroscopy, the molecular details of polysaccharide chain processivity have not been elucidated. Here, we employ simulation to examine how a cellulose chain translocates by a disaccharide unit during the processive cycle of a glycoside hydrolase family 7 cellobiohydrolase. Our results demonstrate that these biologically and industrially important enzymes employ a two-step mechanism for chain threading to form a Michaelis complex and that the free energy barrier to chain threading is significantly lower than the hydrolysis barrier. Taken with previous studies, our findings suggest that the rate-limiting step in enzymatic cellulose degradation is the glycosylation reaction, not chain processivity. Based on the simulations, we find that strong electrostatic interactions with polar residues that are conserved in GH7 cellobiohydrolases, but not in GH7 endoglucanases, at the leading glucosyl ring provide the thermodynamic driving force for polysaccharide chain translocation. Also, we consider the role of aromatic-carbohydrate interactions, which are widespread in carbohydrate-active enzymes and have long been associated with processivity. Our analysis suggests that the primary role for these aromatic residues is to provide tunnel shape and guide the carbohydrate chain to the active site. More broadly, this work elucidates the role of common protein motifs found in carbohydrate-active enzymes that synthesize or depolymerize polysaccharides by chain translocation mechanisms coupled to catalysis.

The mechanism of cellulose hydrolysis by a two-step, retaining cellobiohydrolase elucidated by structural and transition path sampling studies.

Glycoside hydrolases (GHs) cleave glycosidic linkages in carbohydrates, typically via inverting or retaining mechanisms, the latter of which proceeds via a two-step mechanism that includes formation of a glycosyl-enzyme intermediate. We present two new structures of the catalytic domain of Hypocrea jecorina GH Family 7 cellobiohydrolase Cel7A, namely a Michaelis complex with a full cellononaose ligand and a glycosyl-enzyme intermediate, that reveal details of the 'static' reaction coordinate. We also employ transition path sampling to determine the 'dynamic' reaction coordinate for the catalytic cycle. The glycosylation reaction coordinate contains components of forming and breaking bonds and a conformational change in the nucleophile. Deglycosylation proceeds via a product-assisted mechanism wherein the glycosylation product, cellobiose, positions a water molecule for nucleophilic attack on the anomeric carbon of the glycosyl-enzyme intermediate. In concert with previous structures, the present results reveal the complete hydrolytic reaction coordinate for this naturally and industrially important enzyme family.

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.

Homogeneous nucleation of methane hydrates: unrealistic under realistic conditions.

Methane hydrates are ice-like inclusion compounds with importance to the oil and natural gas industry, global climate change, and gas transportation and storage. The molecular mechanism by which these compounds form under conditions relevant to industry and nature remains mysterious. To understand the mechanism of methane hydrate nucleation from supersaturated aqueous solutions, we performed simulations at controlled and realistic supersaturation. We found that critical nuclei are extremely large and that homogeneous nucleation rates are extremely low. Our findings suggest that nucleation of methane hydrates under these realistic conditions cannot occur by a homogeneous mechanism.

A simulation test of the optical Kerr mechanism for laser-induced nucleation.

Recent experiments have demonstrated that intense, nanosecond laser pulses can induce crystal nucleation from supersaturated solutions that are transparent at the incident wavelengths, a phenomenon termed nonphotochemical laser-induced nucleation (NPLIN). Previous work has proposed that this effect is due to the alignment of solute molecules in solution due to the electric field of the applied laser light, promoting crystalline order. We have used simulations of NPLIN to examine how an orientational bias in solution affects nucleation with Monte Carlo simulations of a Potts lattice gas model. We examine this effect within both a classical, one-step nucleation framework as well as in the context of two-step nucleation. Our results indicate that an orientational bias can reduce the free energy barrier to nucleation within the one-step picture as well as promote the crystallization of amorphous precritical nuclei (the rate-determining step in the two-step picture). However, these effects are only present with field strengths that are much greater than those used in experiments.

Communication: Bubbles, crystals, and laser-induced nucleation.

Short intense laser pulses of visible and infrared light can dramatically accelerate crystal nucleation from transparent solutions; previous studies invoke mechanisms that are only applicable for nucleation of ordered phases or high dielectric phases. However, we show that similar laser pulses induce CO(2) bubble nucleation in carbonated water. Additionally, in water that is cosupersaturated with argon and glycine, argon bubbles escaping from the water can induce crystal nucleation without a laser. Our findings suggest a possible link between laser-induced nucleation of bubbles and crystals.

Transition Path Sampling Study of the Feruloyl Esterase Mechanism.

Serine hydrolases cleave peptide and ester bonds and are ubiquitous in nature, with applications in biotechnology, in materials, and as drug targets. The serine hydrolase two-step mechanism employs a serine-histidine-aspartate/glutamate catalytic triad, where the histidine residue acts as a base to activate poor nucleophiles (a serine residue or a water molecule) and as an acid to allow the dissociation of poor leaving groups. This mechanism has been the subject of debate regarding how histidine shuttles the proton from the nucleophile to the leaving group. To elucidate the reaction mechanism of serine hydrolases, we employ quantum mechanics/molecular mechanics-based transition path sampling to obtain the reaction coordinate using the Aspergillus niger feruloyl esterase A (AnFaeA) as a model enzyme. The optimal reaction coordinates include terms involving nucleophilic attack on the carbonyl carbon and proton transfer to, and dissociation of, the leaving group. During the reaction, the histidine residue undergoes a reorientation on the time scale of hundreds of femtoseconds that supports the "moving histidine" mechanism, thus calling into question the "ring flip" mechanism. We find a concerted mechanism, where the transition state coincides with the tetrahedral intermediate with the histidine residue pointed between the nucleophile and the leaving group. Moreover, motions of the catalytic aspartate toward the histidine occur concertedly with proton abstraction by the catalytic histidine and help stabilize the transition state, thus partially explaining how serine hydrolases enable poor nucleophiles to attack the substrate carbonyl carbon. Rate calculations indicate that the second step (deacylation) is rate-determining, with a calculated rate constant of 66 s-1. Overall, these results reveal the pivotal role of active-site dynamics in the catalytic mechanism of AnFaeA, which is likely similar in other serine hydrolases.

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.

Estimating diffusivity along a reaction coordinate in the high friction limit: Insights on pulse times in laser-induced nucleation.

In the high friction limit of Kramers' theory, the diffusion coefficient for motion along the reaction coordinate is a crucial parameter in determining reaction rates from mean first passage times. The Einstein relation between mean squared displacement, time, and diffusivity is inaccurate at short times because of ballistic motion and inaccurate at long times because trajectories drift away from maxima in the potential of mean force. Starting from the Smoluchowski equation for a downward parabolic barrier, we show how drift induced by the potential of mean force can be included in estimating the diffusivity. A modified relation between mean squared displacement, time, and diffusivity now also includes a dependence on the barrier curvature. The new relation provides the diffusivity at the top of the barrier from a linear regression that is analogous to the procedure commonly used with Einstein's relation. The new approach has particular advantages over previous approaches when evaluations of the reaction coordinate are costly or when the reaction coordinate cannot be differentiated to compute restraining forces or velocities. We use the new method to study the dynamics of barrier crossing in a Potts lattice gas model of nucleation from solution. Our analysis shows that some current hypotheses about laser-induced nucleation mechanisms lead to a nonzero threshold laser pulse duration below which a laser pulse will not affect nucleation. We therefore propose experiments that might be used to test these hypotheses.

Engineering enhanced cellobiohydrolase activity.

Glycoside Hydrolase Family 7 cellobiohydrolases (GH7 CBHs) catalyze cellulose depolymerization in cellulolytic eukaryotes, making them key discovery and engineering targets. However, there remains a lack of robust structure-activity relationships for these industrially important cellulases. Here, we compare CBHs from Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A), which exhibit a multi-modular architecture consisting of catalytic domain (CD), carbohydrate-binding module, and linker. We show that PfCel7A exhibits 60% greater performance on biomass than TrCel7A. To understand the contribution of each domain to this improvement, we measure enzymatic activity for a library of CBH chimeras with swapped subdomains, demonstrating that the enhancement is mainly caused by PfCel7A CD. We solve the crystal structure of PfCel7A CD and use this information to create a second library of TrCel7A CD mutants, identifying a TrCel7A double mutant with near-equivalent activity to wild-type PfCel7A. Overall, these results reveal CBH regions that enable targeted activity improvements.