Energetic and mechanistic studies of glucoamylase using molecular recognition of maltose OH groups coupled with site-directed mutagenesis

Molecular recognition using a series of deoxygenated maltose analogues was used to determine the substrate transition-state binding energy profiles of 10 single-residue mutants at the active site of glucoamylase from Aspergillus niger. The individual contribution of each substrate hydroxyl group to transition-state stabilization with the wild type and each mutant GA was determined from the relation Delta(DeltaG()) = -RT ln[(k(cat)/K(M))(x)/(k(cat)/K(M))(y)], where x represents either a mutant enzyme or substrate analogue and y the wild-type enzyme or parent substrate. The resulting binding energy profiles indicate that disrupting an active site hydrogen bond between enzyme and substrate, as identified in crystal structures, not only sharply reduces or eliminates the energy contributed from that particular hydrogen bond but also perturbs binding contributions from other substrate hydroxyl groups. Replacing the active site acidic groups, Asp55, Glu180, or Asp309, with the corresponding amides, and the neutral Trp178 with the basic Arg, all substantially reduced the binding energy contribution of the 4'- and 6'-OH groups of maltose at subsite -1, even though both Glu180 and Asp309 are localized at subsite 1. In contrast, the substitution, Asp176 --> Asn, located near subsites -1 and 1, did not substantially perturb any of the individual hydroxyl group binding energies. Similarly, the substitutions Tyr116 --> Ala, Ser119 --> Tyr, or Trp120 --> Phe also did not substantially alter the energy profiles even though Trp120 has a critical role in directing conformational changes necessary for activity. Since the mutations at Trp120 and Asp176 reduced k(cat) values by 50- and 12-fold, respectively, a large effect on k(cat) is not necessarily accompanied by changes in hydroxyl group binding energy contributions. Two substitutions, Asn182 --> Ala and Tyr306 --> Phe, had significant though small effects on interactions with 3- and 4'-OH, respectively. Binding interactions between the enzyme and the glucosyl group in subsite -1, particularly with the 4'- and 6'-OH groups, play an important role in substrate binding, while subsite 1 interactions may play a more important role in product release.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Automated docking of alpha-(1-->4)- and alpha-(1-->6)-linked glucosyl trisaccharides and maltopentaose into the soybean beta-amylase active site

The Lamarckian genetic algorithm of AutoDock 3.0 was used to dock alpha-maltotriose, methyl alpha-panoside, methyl alpha-isopanoside, methyl alpha-isomaltotrioside, methyl alpha-(6(1)-alpha-glucopyranosyl)-maltoside, and alpha-maltopentaose into the closed and, except for alpha-maltopentaose, into the open conformation of the soybean beta-amylase active site. In the closed conformation, the hinged flap at the mouth of the active site closes over the substrate. The nonreducing end of alpha-maltotriose docks preferentially to subsites -2 or +1, the latter yielding nonproductive binding. Some ligands dock into less optimal conformations with the nonreducing end at subsite -1. The reducing-end glucosyl residue of nonproductively-bound alpha-maltotriose is close to residue Gln194, which likely contributes to binding to subsite +3. In the open conformation, the substrate hydrogen-bonds with several residues of the open flap. When the flap closes, the substrate productively docks if the nonreducing end is near subsites -2 or -1. Trisaccharides with alpha-(1-->6) bonds do not successfully dock except for methyl alpha-isopanoside, whose first and second glucosyl rings dock exceptionally well into subsites -2 and -1. The alpha-(1-->6) bond between the second and third glucosyl units causes the latter to be improperly positioned into subsite +1; the fact that isopanose is not a substrate of beta-amylase indicates that binding to this subsite is critical for hydrolysis.     

Dissecting the catalytic mechanism of a plant beta-D-glucan glucohydrolase through structural biology using inhibitors and substrate analogues

Higher plant, family GH3 beta-D-glucan glucohydrolases exhibit exo-hydrolytic and retaining (e-->e) mechanisms of action and catalyze the removal of single glucosyl residues from the non-reducing termini of beta-D-linked glucosidic substrates, with retention of anomeric configuration. The broad specificity beta-D-glucan glucohydrolases are likely to play roles in cell wall re-modelling, turn-over of cell wall components and possibly in plant defence reactions against pathogens. Crystal structures of the barley beta-D-glucan glucohydrolase, obtained from both native enzyme and from the enzyme in complex with a substrate analogues and mechanism-based inhibitors, have enabled the basis of substrate specificity, the mechanism of catalysis, and the role of domain movements during the catalytic cycle to be defined in precise molecular terms. The active site of the enzyme forms a shallow 'pocket' that is located at the interface of two domains of the enzyme and accommodates two glucosyl residues. The propensity of the enzyme to hydrolyze a broad range of substrates with (1-->2)-, (1-->3)-, (1-->4)- and (1-->6)-beta-D-glucosidic linkages is explained from crystal structures of the enzyme in complex with non-hydrolysable S-glycoside substrate analogues, and from molecular modelling. During binding of gluco-oligosaccharides, the glucosyl residue at subsite -1 is locked in a highly constrained position, but the glucosyl residue at the +1 subsite is free to adjust its position between two tryptophan residues positioned at the entry of the active site pocket. The flexibility at subsite +1 and the projection of the remainder of the substrate away from the pocket provide a structural rationale for the capacity of the enzyme to accommodate and hydrolyze glucosides with different linkage positions and hence different overall conformations. While mechanism-based inhibitors with micromolar Ki constants bind in the active site of the enzyme and form esters with the catalytic nucleophile, transition-state mimics bind with their 'glucose' moieties distorted into the 4E conformation, which is critical for the nanomolar binding of these inhibitors to the enzyme. The glucose product of the reaction, which is released from the non-reducing termini of substrates, remains bound to the beta-D-glucan glucohydrolase in the -1 subsite of the active site, until a new substrate molecule approaches the enzyme. If dissociation of the glucose from the enzyme active site could be synchronized throughout the crystal, time-resolved Laue X-ray crystallography could be used to follow the conformational changes that occur as the glucose product diffuses away and the incoming substrate is bound by the enzyme.     

Insights into ligand-induced conformational change in Cel5A from Bacillus agaradhaerens revealed by a catalytically active crystal form

Glycoside hydrolases are ubiquitous enzymes involved in a diverse array of biological processes, from the breakdown of biomass, through to viral invasion and cellular signalling. Endoglucanase Cel5A from Bacillus agaradhaerens, classified into glycoside hydrolase family 5, has been studied in a catalytically inactive crystal form at low pH conditions, in which native and complex structures revealed the importance of ring distortion during catalysis. Here, we present the structure of Cel5A in a new crystal form obtained at higher pH values in which the enzyme is active "in-crystal". Native, cellotriosyl-enzyme intermediate and beta-d-cellobiose structures were solved at 1.95, 1.75 and 2.1 A resolution, respectively. These structures reveal two classes of conformational change: those caused by crystal-packing and pH, with others induced upon substrate binding. At pH 7 a histidine residue, His206, implicated in substrate-binding and catalysis, but previously far removed from the substrate-binding cleft, moves over 10 A into the active site cleft in order to interact with the substrate in the +2 subsite. Occupation of the -1 subsite by substrate induces a loop closure to optimise protein-ligand interactions. Cel5A, along with the unrelated family 45 and family 6 cellulases, provides further evidence of substantial conformational change in response to ligand binding for this class of hydrolytic enzyme.     

Crystal complex structures reveal how substrate is bound in the -4 to the +2 binding sites of Humicola grisea Cel12A

As part of an ongoing enzyme discovery program to investigate the properties and catalytic mechanism of glycoside hydrolase family 12 (GH 12) endoglucanases, a GH family that contains several cellulases that are of interest in industrial applications, we have solved four new crystal structures of wild-type Humicola grisea Cel12A in complexes formed by soaking with cellobiose, cellotetraose, cellopentaose, and a thio-linked cellotetraose derivative (G2SG2). These complex structures allow mapping of the non-covalent interactions between the enzyme and the glucosyl chain bound in subsites -4 to +2 of the enzyme, and shed light on the mechanism and function of GH 12 cellulases. The unhydrolysed cellopentaose and the G2SG2 cello-oligomers span the active site of the catalytically active H.grisea Cel12A enzyme, with the pyranoside bound in subsite -1 displaying a S31 skew boat conformation. After soaking in cellotetraose, the cello-oligomer that is found bound in site -4 to -1 contains a beta-1,3-linkage between the two cellobiose units in the oligomer, which is believed to have been formed by a transglycosylation reaction that has occurred during the ligand soak of the protein crystals. The close fit of this ligand and the binding sites occupied suggest a novel mixed beta-glucanase activity for this enzyme.     

Interaction of substrate uridyl 3'',5''-adenosine with ribonuclease A: a molecular dynamics study.

A wealth of information available from x-ray crystallographic structures of enzyme-ligand complexes makes it possible to study interactions at the molecular level. However, further investigation is needed when i) the binding of the natural substrate must be characterized, because ligands in the stable enzyme-ligand complexes are generally inhibitors or the analogs of substrate and transition state, and when ii) ligand binding is in part poorly characterized. We have investigated these aspects in the binding of substrate uridyl 3',5'-adenosine (UpA) to ribonuclease A (RNase A). Based on the systematically docked RNase A-UpA complex resulting from our previous study, we have undertaken a molecular dynamics simulation of the complex with solvent molecules. The molecular dynamics trajectories of this complex are analyzed to provide structural explanations for varied experimental observations on the ligand binding at the B2 subsite of ribonuclease A. The present study suggests that B2 subsite stabilization can be effected by different active site groups, depending on the substrate conformation. Thus when adenosine ribose pucker is O4'-endo, Gln69 and Glu111 form hydrogen-bonding contacts with adenine base, and when it is C2'-endo, Asn71 is the only amino acid residue in direct contact with this base. The latter observation is in support of previous mutagenesis and kinetics studies. Possible roles for the solvent molecules in the binding subsites are described. Furthermore, the substrate conformation is also examined along the simulation pathway to see if any conformer has the properties of a transition state. This study has also helped us to recognize that small but concerted changes in the conformation of the substrate can result in substrate geometry favorable for 2',3' cyclization. The identified geometry is suitable for intraligand proton transfer between 2'-hydroxyl and phosphate oxygen atom. The possibility of intraligand proton transfer as suggested previously and the mode of transfer before the formation of cyclic intermediate during transphosphorylation are discussed.     

Crystallographic evidence for substrate ring distortion and protein conformational changes during catalysis in cellobiohydrolase Ce16A from trichoderma reesei.

BACKGROUND: Cel6A is one of the two cellobiohydrolases produced by Trichoderma reesei. The catalytic core has a structure that is a variation of the classic TIM barrel. The active site is located inside a tunnel, the roof of which is formed mainly by a pair of loops. RESULTS: We describe three new ligand complexes. One is the structure of the wild-type enzyme in complex with a nonhydrolysable cello-oligosaccharide, methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside (Glc)(2)-S-(Glc)(2), which differs from a cellotetraose in the nature of the central glycosidic linkage where a sulphur atom replaces an oxygen atom. The second structure is a mutant, Y169F, in complex with the same ligand, and the third is the wild-type enzyme in complex with m-iodobenzyl beta-D-glucopyranosyl-beta(1,4)-D-xylopyranoside (IBXG). CONCLUSIONS: The (Glc)(2)-S-(Glc)(2) ligand binds in the -2 to +2 sites in both the wild-type and mutant enzymes. The glucosyl unit in the -1 site is distorted from the usual chair conformation in both structures. The IBXG ligand binds in the -2 to +1 sites, with the xylosyl unit in the -1 site where it adopts the energetically favourable chair conformation. The -1 site glucosyl of the (Glc)(2)-S-(Glc)(2) ligand is unable to take on this conformation because of steric clashes with the protein. The crystallographic results show that one of the tunnel-forming loops in Cel6A is sensitive to modifications at the active site, and is able to take on a number of different conformations. One of the conformational changes disrupts a set of interactions at the active site that we propose is an integral part of the reaction mechanism.     

Structural basis for broad substrate specificity in higher plant beta-D-glucan glucohydrolases

Family 3 beta-D-glucan glucohydrolases are distributed widely in higher plants. The enzymes catalyze the hydrolytic removal of beta-D-glucosyl residues from nonreducing termini of a range of beta-D-glucans and beta-D-oligoglucosides. Their broad specificity can be explained by x-ray crystallographic data obtained from a barley beta-D-glucan glucohydrolase in complex with nonhydrolyzable S-glycoside substrate analogs and by molecular modeling of enzyme/substrate complexes. The glucosyl residue that occupies binding subsite -1 is locked tightly into a fixed position through extensive hydrogen bonding with six amino acid residues near the bottom of an active site pocket. In contrast, the glucosyl residue at subsite +1 is located between two Trp residues at the entrance of the pocket, where it is constrained less tightly. The relative flexibility of binding at subsite +1, coupled with the projection of the remainder of bound substrate away from the enzyme's surface, means that the overall active site can accommodate a range of substrates with variable spatial dispositions of adjacent beta-D-glucosyl residues. The broad specificity for glycosidic linkage type enables the enzyme to perform diverse functions during plant development.     

Circular dichroic evidence for an ordered sequence of ligand/binding site interactions in the catalytic reaction of the cAMP-dependent protein kinase

A limiting requirement for substrate specificity of the cAMP-dependent protein kinase is the presence of one or two basic residues located to the N-terminal side of the target substrate serine. Furthermore, circular dichroic (CD) studies have shown that binding of protein substrate involves a series of at least two independent conformational changes in the enzyme, each of which is initiated by a recognition signal on the substrate protein. The present study attempts to elucidate further the complete sequence of enzyme/ligand interactions by using the synthetic substrate peptide Kemptide and analogues differing from it at crucial points in the sequence: the Ala-peptide, where alanine is substituted for the target serine, and D-Ser-Kemptide, where the target serine is in the D rather than the L configuration. Examination of the effects of binding of these substrates on the intrinsic UV CD of the enzyme and the induced CD in the presence of Blue Dextran has revealed a third step in the substrate/enzyme binding interaction. Although sections of the conformational change at the active site are dependent on the basic subsite and the serine hydroxyl group on the peptide, respectively, the complete conformational change requires that the substrate be bound in random coil conformation. Where this does not occur, the kinetics show that the peptide will not act either as substrate or as inhibitor of the enzyme. Further, the interaction between the serine hydroxyl group and an enzyme tyrosine residue, previously observed, appears to be dependent on the correct orientation as well as the mere presence of the target -OH group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic (0.94 A) resolution structure of an inverting glycosidase in complex with substrate

The crystal structure of Clostridium thermocellum endoglucanase CelA in complex with cellopentaose has been determined at 0.94 A resolution. The oligosaccharide occupies six D-glucosyl-binding subsites, three on either side of the scissile glycosidic linkage. The substrate and product of the reaction occupy different positions at the reducing end of the cleft, where an extended array of hydrogen-bonding interactions with water molecules fosters the departure of the leaving group. Severe torsional strain upon the bound substrate forces a distorted boat(2,5) B conformation for the glucosyl residue bound at subsite -1, which facilitates the formation of an oxocarbenium ion intermediate and might favor the breakage of the sugar ring concomitant with catalysis.     

Crystal structure of Thermobifida fusca endoglucanase Cel6A in complex with substrate and inhibitor: the role of tyrosine Y73 in substrate ring distortion

Endoglucanase Cel6A from Thermobifida fusca hydrolyzes the beta-1,4 linkages in cellulose at accessible points along the polymer. The structure of the catalytic domain of Cel6A from T. fusca in complex with a nonhydrolysable substrate analogue that acts as an inhibitor, methylcellobiosyl-4-thio-beta-cellobioside (Glc(2)-S-Glc(2)), has been determined to 1.5 A resolution. The glycosyl unit in subsite -1 was sterically hindered by Tyr73 and forced into a distorted (2)S(o) conformation. In the enzyme where Tyr73 was mutated to a serine residue, the hindrance was removed and the glycosyl unit in subsite -1 had a relaxed (4)C(1) chair conformation. The relaxed conformation was seen in two complex structures of the mutated enzyme, with cellotetrose (Glc(4)) at 1.64 A and Glc(2)-S-Glc(2) at 1.04 A resolution.     

Effects of noncatalytic residue mutations on substrate specificity and ligand binding of Thermobifida fusca endocellulase cel6A.

The availability of a high-resolution structure of the Thermobifida fusca endocellulase Cel6A catalytic domain makes this enzyme ideal for structure-based efforts to engineer cellulases with high activity on native cellulose. In order to determine the role of conserved, noncatalytic residues in cellulose hydrolysis, 14 mutations of six conserved residues in or near the Cel6A active-site cleft were studied for their effects on catalytic activity, substrate specificity, processivity and ligand-binding affinity. Eleven mutations were generated by site-directed mutagenesis using PCR, while three were from previous studies. All the CD spectra of the mutant enzymes were indistinguishable from that of Cel6A indicating that the mutations did not dramatically change protein conformation. Seven mutations in four residues (H159, R237, K259 and E263) increased activity on carboxymethyl cellulose (CM-cellulose), with K259H (in glucosyl subsite -2) creating the highest activity (370%). Interestingly, the other mutations in these residues reduced CM-cellulose activity. Only the K259H enzyme retained more activity on acid-swollen cellulose than on filter paper, suggesting that this mutation affected the rate-limiting step in crystalline cellulose hydrolysis. All the mutations lowered activity on cellotriose and cellotetraose, but two mutations, both in subsite +1 (H159S and N190A), had higher kcat/Km values (6.6-fold and 5.0-fold, respectively) than Cel6A on 2,4-dinitrophenyl-beta-D-cellobioside. Measurement of enzyme : ligand dissociation constants for three methylumbelliferyl oligosaccharides and cellotriose showed that all mutant enzymes bound these ligands either to the same extent as or more weakly than Cel6A. These results show that conserved noncatalytic residues can profoundly affect Cel6A activity and specificity.     

Crystal structures of amylosucrase from Neisseria polysaccharea in complex with D-glucose and the active site mutant Glu328Gln in complex with the natural substrate sucrose

The structure of amylosucrase from Neisseria polysaccharea in complex with beta-D-glucose has been determined by X-ray crystallography at a resolution of 1.66 A. Additionally, the structure of the inactive active site mutant Glu328Gln in complex with sucrose has been determined to a resolution of 2.0 A. The D-glucose complex shows two well-defined D-glucose molecules, one that binds very strongly in the bottom of a pocket that contains the proposed catalytic residues (at the subsite -1), in a nonstrained (4)C(1) conformation, and one that binds in the packing interface to a symmetry-related molecule. A third weaker D-glucose-binding site is located at the surface near the active site pocket entrance. The orientation of the D-glucose in the active site emphasizes the Glu328 role as the general acid/base. The binary sucrose complex shows one molecule bound in the active site, where the glucosyl moiety is located at the alpha-amylase -1 position and the fructosyl ring occupies subsite +1. Sucrose effectively blocks the only visible access channel to the active site. From analysis of the complex it appears that sucrose binding is primarily obtained through enzyme interactions with the glucosyl ring and that an important part of the enzyme function is a precise alignment of a lone pair of the linking O1 oxygen for hydrogen bond interaction with Glu328. The sucrose specificity appears to be determined primarily by residues Asp144, Asp394, Arg446, and Arg509. Both Asp394 and Arg446 are located in an insert connecting beta-strand 7 and alpha-helix 7 that is much longer in amylosucrase compared to other enzymes from the alpha-amylase family (family 13 of the glycoside hydrolases).     

Differences in active site structure in a family of beta-glucan endohydrolases deduced from the kinetics of inactivation by epoxyalkyl beta-oligoglucosides

The active sites of a spectrum of beta-glucan endohydrolases with distinct, but related substrate specificities have been probed using a series of epoxyalkyl beta-glycosides of glucose, cellobiose, cellotriose, laminaribiose, laminaritriose, 3O-beta-D-glucosyl-cellobiose and 4O-beta-D-glucosyl-laminaribiose with different aglycon chain lengths. The inactivation of each of the endohydrolases by these compounds results from active site-directed inhibitor action, as indicated by the dependence of the inactivation rate on pH, glycosyl chain length and linkage position, aglycon length, and the protective effect of disaccharides derived from the natural substrates. Comparisons of inhibitor specificity between a Bacillus subtilis 1,3;1,4-beta-D-glucan 4-glucanohydrolase (EC 3.2.1.73), a Streptomyces cellulase (EC 3.2.1.4), a Schizophyllum commune cellulase (EC 3.2.1.4), a Rhizopus arrhizus 1,3-(1,3;1,4)-beta-D-glucan 3(4)-glucanohydrolase (EC 3.2.1.6), and a Nicotiana glutinosa 1,3-beta-D-glucan 3-glucanohydrolase (EC 3.2.1.39) demonstrated different tolerances for glycosyl linkage positions in the inactivation process and a critical role of aglycon length reflecting differences in the active site geometry of the enzymes. For the B. subtilis endohydrolase it was concluded that the aglycon residue of the inhibitor spans the glycosyl binding subsite occupied by the 3-substituted glucosyl residue involved in the glucosidic linkage cleaved in the natural substrate. Appropriate positioning of the inhibitor epoxide group with respect to the catalytic amino acids in the active site is crucial to the inactivation step and the number of glucosyl residues in the inhibitor affects aglycon chain length specificity. The importance of this effect differs between the glucanases tested and may be related to the number of glycosyl binding subsites in the active site.     

Role of Trp140 at subsite -6 on the maltohexaose production of maltohexaose-producing amylase from alkalophilic Bacillus sp.707

Maltohexaose-producing amylase (G6-amylase) from alkalophilic Bacillus sp.707 predominantly produces maltohexaose (G6) in the yield of >30% of the total products from short-chain amylose (DP=17). Our previous crystallographic study showed that G6-amylase has nine subsites, from -6 to +3, and pointed out the importance of the indole moiety of Trp140 in G6 production. G6-amylase has very low levels of hydrolytic activities for oligosaccharides shorter than maltoheptaose. To elucidate the mechanism underlying G6 production, we determined the crystal structures of the G6-amylase complexes with G6 and maltopentaose (G5). In the active site of the G6-amylase/G5 complex, G5 is bound to subsites -6 to -2, while G1 and G6 are found at subsites +2 and -7 to -2, respectively, in the G6-amylase/G6 complex. In both structures, the glucosyl residue located at subsite -6 is stacked to the indole moiety of Trp140 within a distance of 4A. The measurement of the activities of the mutant enzymes when Trp140 was replaced by leucine (W140L) or by tyrosine (W140Y) showed that the G6 production from short-chain amylose by W140L is lower than that by W140Y or wild-type enzyme. The face-to-face short contact between Trp140 and substrate sugars is suggested to regulate the disposition of the glucosyl residue at subsite -6 and to govern product specificity for G6 production.     

Molecular dynamics simulation of the processive endocellulase Cel48F from Clostridium cellulolyticum: a novel “water‐control mechanism” in enzymatic hydrolysis of cellulose

Glycoside hydrolase of Cel48F from Clostridium cellulolyticum is an important processive cellulose, which can hydrolyze cellulose into cellobiose. Molecular dynamics simulations were used to investigate the hydrolysis mechanism of cellulose. The two conformations of the Cel48F‐cellotetrose complex in which the cellotetroses are bound at different sites (known as the sliding conformation and the hydrolyzing conformation) were simulated. By comparing these two conformations, a water‐control mechanism is proposed, in which the hydrolysis proceeds by providing a water molecule for every other glucosidic linkage. The roles of certain key residues are determined: Glu55 and Asp230 are the most probable candidates for acid and base, respectively, in the mechanism of inverting anomeric carbon. Met414 and Trp417 constitute the water‐control system. Glu44 might keep the substrate at a certain location within the active site or help the substrate chain to move from the sliding conformation to the hydrolyzing conformation. The other hydrophobic residues around the substrate can decrease the sliding energy barrier or provide a hydrophobic environment to resist entry of the surrounding water molecules into the active site, except for those coming from a specific water channel. Copyright © 2014 John Wiley & Sons, Ltd.     

Automated docking to explore subsite binding by glycoside hydrolase family 6 cellobiohydrolases and endoglucanases

Cellooligosaccharides were computationally docked using AutoDock into the active sites of the glycoside hydrolase Family 6 enzymes Hypocrea jecorina (formerly Trichoderma reesei) cellobiohydrolase and Thermobifida fusca endoglucanase. Subsite -2 exerts the greatest intermolecular energy in binding beta-glucosyl residues, with energies progressively decreasing to either side. Cumulative forces imparting processivity exerted by these two enzymes are significantly less than by the equivalent glycoside hydrolase Family 7 enzymes studied previously. Putative subsites -4, -3, +3, and +4 exist in H. jecorina cellobiohydrolase, along with putative subsites -4, -3, and +3 in T. fusca endoglucanase, but they are less important than subsites -2, -1, +1, and +2. In general, binding adds 3-7 kcal/mol to ligand intramolecular energies because of twisting of scissile glycosidic bonds. Distortion of beta-glucosyl residues to the (2)S(O) conformation by binding in subsite -1 adds approximately 7 kcal/mol to substrate intramolecular energies.     

Structure and dynamics of Trichoderma harzianum Cel7B suggest molecular architecture adaptations required for a wide spectrum of activities on plant cell wall polysaccharides

Cellulases from glycoside hydrolase family 7 (GH7) play crucial roles in plant lignocellulose deconstruction by fungi, but structural information available for GH7 fungal endoglucanases is limited when compared to the number of known sequences in the family. Here, we report the X-ray structure of the glycosylated catalytic domain (CD) of Trichoderma harzianum endoglucanase, ThCel7B, solved and refined at 2.9 Å resolution. Additionally, our extensive molecular dynamics simulations of this enzyme in complex with a variety of oligosaccharides provide a better understanding of its promiscuous hydrolytic activities on plant cell wall polysaccharides. The simulations demonstrate the importance of the hydrogen bond between substrate O2 hydroxyl in the subsite −1 and a side chain of catalytic Glu196 which renders ThCel7B capable to catalytically cleave cello and xylooligosaccharides, but not mannooligosaccharides. Moreover, detailed structural analyses and MD simulations revealed an additional binding pocket, suitable for accommodation of oligosaccharide decorations and/or substrates with mixed glycoside bonds that abuts onto the binding cleft close to subsite +2.