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.

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.

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.

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.

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 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.

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.

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.

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.

Simulations of cellulose translocation in the bacterial cellulose synthase suggest a regulatory mechanism for the dimeric structure of cellulose.

The processive cycle of the bacterial cellulose synthase (Bcs) includes the addition of a single glucose moiety to the end of a growing cellulose chain followed by the translocation of the nascent chain across the plasma membrane. The mechanism of this translocation and its precise location within the processive cycle are not well understood. In particular, the molecular details of how a polymer (cellulose) whose basic structural unit is a dimer (cellobiose) can be constructed by adding one monomer (glucose) at a time are yet to be elucidated. Here, we have utilized molecular dynamics simulations and free energy calculations to the shed light on these questions. We find that translocation forward by one glucose unit is quite favorable energetically, giving a free energy stabilization of greater than 10 kcal/mol. In addition, there is only a small barrier to translocation, implying that translocation is not rate limiting within the Bcs processive cycle (given experimental rates for cellulose synthesis in vitro). Perhaps most significantly, our results also indicate that steric constraints at the transmembrane tunnel entrance regulate the dimeric structure of cellulose. Namely, when a glucose molecule is added to the cellulose chain in the same orientation as the acceptor glucose, the terminal glucose freely rotates upon forward motion, thus suggesting a regulatory mechanism for the dimeric structure of cellulose. We characterize both the conserved and non-conserved enzyme-polysaccharide interactions that drive translocation, and find that 20 of the 25 residues that strongly interact with the translocating cellulose chain in the simulations are well conserved, mostly with polar or aromatic side chains. Our results also allow for a dynamical analysis of the role of the so-called `finger helix' in cellulose translocation that has been observed structurally. Taken together, these findings aid in the elucidation of the translocation steps of the Bcs processive cycle and may be widely relevant to polysaccharide synthesizing or degrading enzymes that couple catalysis with chain translocation.

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.

Who's on base? Revealing the catalytic mechanism of inverting family 6 glycoside hydrolases.

In several important classes of inverting carbohydrate-active enzymes, the identity of the catalytic base remains elusive, including in family 6 Glycoside Hydrolase (GH6) enzymes, which are key components of cellulase cocktails for cellulose depolymerization. Despite many structural and kinetic studies with both wild-type and mutant enzymes, especially on the Trichoderma reesei (Hypocrea jecorina) GH6 cellulase (TrCel6A), the catalytic base in the single displacement inverting mechanism has not been definitively identified in the GH6 family. Here, we employ transition path sampling to gain insight into the catalytic mechanism, which provides unbiased atomic-level understanding of key order parameters involved in cleaving the strong glycosidic bond. Our hybrid quantum mechanics and molecular mechanics (QM/MM) simulations reveal a network of hydrogen bonding that aligns two active site water molecules that play key roles in hydrolysis: one water molecule drives the reaction by nucleophilic attack on the substrate and a second shuttles a proton to the putative base (D175) via a short water wire. We also investigated the case where the putative base is mutated to an alanine, an enzyme that is experimentally still partially active. The simulations predict that proton hopping along a water wire via a Grotthuss mechanism provides a mechanism of catalytic rescue. Further simulations reveal that substrate processive motion is 'driven' by strong electrostatic interactions with the protein at the product sites and that the -1 sugar adopts a 2SO ring configuration as it reaches its binding site. This work thus elucidates previously elusive steps in the processive catalytic mechanism of this important class of enzymes.

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.

Reaction coordinate of incipient methane clathrate hydrate nucleation.

Nucleation from solution is a ubiquitous phenomenon with relevance to myriad scientific disciplines, including pharmaceuticals, biomineralization, and disease. One prominent example is the nucleation of clathrate hydrates, multicomponent crystalline inclusion compounds relevant to the energy industry where they block pipelines and also constitute a potential vast energy resource. Despite their importance, the molecular mechanism of incipient hydrate formation remains unknown. Herein, we employ advanced molecular simulation tools (pB histogram, equilibrium path sampling) to provide a statistical-mechanical basis for extracting physical insight into the molecular steps by which clathrates form. Through testing the Mutually Coordinated Guest (MCG) order parameter, we demonstrate that both guest (methane) and host (water) structuring are crucial to accurately describe the nucleation of hydrates and determine a critical nucleus size of MCG-1 = 16 at 255 K and 500 bar. Equipped with a validated (and novel) reaction coordinate, subsequent equilibrium path sampling simulations yield the free energy barrier and nucleation rate. The resulting quantitative nucleation process is described by the MCG clustering mechanism. This constitutes a significant advance in the field of hydrates research, as the fitness of a molecular descriptor has never been statistically verified. More broadly, this work has significance to a wide range of multicomponent nucleation contexts wherein the formation mechanism depends on contributions from both solute and solvent.

Towards a molecular-level theory of carbohydrate processivity in glycoside hydrolases.

Polysaccharide depolymerization in nature is primarily accomplished by processive glycoside hydrolases (GHs), which abstract single carbohydrate chains from polymer crystals and cleave glycosidic linkages without dissociating after each catalytic event. Understanding the molecular-level features and structural aspects of processivity is of importance due to the prevalence of processive GHs in biomass-degrading enzyme cocktails. Here, we describe recent advances towards the development of a molecular-level theory of processivity for cellulolytic and chitinolytic enzymes, including the development of novel methods for measuring rates of key steps in processive action and insights gained from structural and computational studies. Overall, we present a framework for developing structure-function relationships in processive GHs and outline additional progress towards developing a fundamental understanding of these industrially important enzymes.

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.