Force calculations in automated docking: enzyme-substrate interactions in Fusarium oxysporum Cel7B

AutoDock is a small-molecule docking program that uses an energy function to score docked ligands. Here AutoDock's grid-based method for energy evaluation was exploited to evaluate the force exerted by Fusarium oxysporum Cel7B on the atoms of docked cellooligosaccharides and a thiooligosaccharide substrate analog. Coupled with the interaction energies evaluated for each docked ligand, these forces give insight into the dynamics of the ligand in the active site, and help to elucidate the relative importance of specific enzyme-substrate interactions in stabilizing the substrate transition-state conformation. The processive force on the docked substrate in the F. oxysporum Cel7B active site is less than half of that on the docked substrate in the Hypocrea jecorina Cel7A active site. Hydrogen bonding interactions of the enzyme with the C2 hydroxyl group of the glucosyl residue in subsite -2 and with the C3 hydroxyl group of the glucosyl residue in subsite +1 are the most significant in stabilizing the distorted14B transition-state conformation of the glucosyl residue in subsite -1. The force calculations also help to elucidate the mechanism that prevents the active site from fouling.     

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.     

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.     

Structural basis for ligand binding and processivity in cellobiohydrolase Cel6A from Humicola insolens

The enzymatic digestion of cellulose entails intimate involvement of cellobiohydrolases, whose characteristic active-center tunnel contributes to a processive degradation of the polysaccharide. The cellobiohydrolase Cel6A displays an active site within a tunnel formed by two extended loops, which are known to open and close in response to ligand binding. Here we present five structures of wild-type and mutant forms of Cel6A from Humicola insolens in complex with nonhydrolyzable thio-oligosaccharides, at resolutions from 1.7-1.1 A, dissecting the structural accommodation of a processing substrate chain through the active center during hydrolysis. Movement of ligand is facilitated by extensive solvent-mediated interactions and through flexibility in the hydrophobic surfaces provided by a sheath of tryptophan residues.     

Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var. mycoides cocrystallized with maltopentaose

The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state.     

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).     

New ganglioside analogs that inhibit influenza virus sialidase

Synthetic thioglycoside-analogs of gangliosides such as Neu5Ac alpha(2-S-6)Glc beta(1-1)Ceramide (1) and the GM3 analog Neu5Ac alpha(2-S-6)Gal beta(1-4)Glc beta(1-1)Ceramide (2), competitively inhibited GM3 hydrolysis by the sialidase of different subtypes of human and animal influenza viruses with an apparent Ki value of 2.8 x 10(-6) and 1.5 x 10(-5) M, respectively. The inhibitory activity of the ganglioside GM4 analog [Neu5Ac alpha(2-S-6)Gal beta(1-1)Ceramide (3)], in which the glucose of 1 was substituted by galactose, was lower than that of 1 (Ki = 1.0 x 10(-4) M). The thioglycoside-analogs (1, 2, 3) of the gangliosides were non-hydrolyzable substrates for influenza virus sialidase. The inhibitory activity of 1 to bacterial sialidases from Clostridium perfringens and Arthrobacter ureafaciens was considerably lower than that to influenza virus sialidase, indicating that the structure of the active site in bacterial and influenza virus sialidase may be different and the analogs may be useful to determine the orientation of the substrate to the active site of sialidases, especially of influenza viruses.     

Structural basis for enantiomer binding and separation of a common beta-blocker: crystal structure of cellobiohydrolase Cel7A with bound (S)-propranolol at 1.9 A resolution

Cellobiohydrolase Cel7A (previously called CBH 1), the major cellulase produced by the mould fungus Trichoderma reesei, has been successfully exploited as a chiral selector for separation of stereo-isomers of some important pharmaceutical compounds, e.g. adrenergic beta-blockers. Previous investigations, including experiments with catalytically deficient mutants of Cel7A, point unanimously to the active site as being responsible for discrimination of enantiomers.In this work the structural basis for enantioselectivity of basic drugs by Cel7A has been studied by X-ray crystallography. The catalytic domain of Cel7A was co-crystallised with the (S)-enantiomer of a common beta-blocker, propranolol, at pH 7, and the structure of the complex was determined and refined at 1. 9 A resolution. Indeed, (S)-propranolol binds at the active site, in glucosyl-binding subsites -1/+1. The catalytic residues Glu212 and Glu217 make tight salt links with the secondary amino group of (S)-propranolol. The oxygen atom attached to the chiral centre of (S)-propranolol forms hydrogen bonds to the nucleophile Glu212 O(epsilon1) and to Gln175 N(epsilon2), whereas the aromatic naphthyl moiety stacks with the indole ring of Trp376 in site +1. The bidentate charge interaction with the catalytic glutamate residues is apparently crucial, since no enantioselectivity has been obtained with the catalytically deficient mutants E212Q and E217Q.Activity inhibition experiments with wild-type Cel7A were performed in conditions close to those used for crystallisation. Competitive inhibition constants for (R)- and (S)-propranolol were determined at 220 microM and 44 microM, respectively, corresponding to binding free energies of 20 kJ/mol and 24 kJ/mol, respectively. The K(i) value for (R)-propranolol was 57-fold lower than the highest concentration, 12.5 mM, used in co-crystallisation experiments. Still several attempts to obtain a complex with the (R)-enantiomer have failed.By using cellobiose as a selective competing ligand, the retention of the enantiomers of propranolol on the chiral stationary phase (CSP) based on Cel7A mutant D214N were resolved into enantioselective and non- selective binding. The enantioselective binding was weaker for both enantiomers on D214N-CSP than on wild-type-CSP.     

Binding of enzyme IIAGlc, a component of the phosphoenolpyruvate:sugar phosphotransferase system, to the Escherichia coli lactose permease

Enzyme IIA(Glc), encoded by the crr gene of the phosphoenolpyruvate:sugar phosphotransferase system, plays an important role in regulating intermediary metabolism in Escherichia coli ("catabolite repression"). One function involves inhibition of inducible transport systems ("inducer exclusion"), and with lactose permease, a galactoside is required for unphosphorylated IIA(Glc) binding to cytoplasmic loops IV/V and VI/VII [Sondej, M., Sun, J. et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 3525-3530]. With inside-out membrane vesicles containing the permease, [(125)I]IIA(Glc) binding promoted by melibiose exhibits an affinity (K(D)(IIA)) of approximately 1 microM and a stoichiometry of one mole of IIA(Glc) per six moles of lactose permease. Both the quantity of [(125)I]IIA(Glc) bound and the sugar concentration required for half-maximal IIA(Glc) binding (K(0.5)(IIA)(sug)) was measured for eight permease substrates. Differences in maximal IIA(Glc) binding are observed, and the K(0.5)(IIA)(sug) does not correlate with the affinity of LacY for sugar. Furthermore, K(0.5)(IIA)(sug) does not correlate with sugar affinities for various permease mutants. IIA(Glc) does not bind to a mutant (Cys154 --> Gly), which is locked in an outwardly facing conformation, binds with increased stoichiometry to mutant Lys131 --> Cys, and binds only weakly to two other mutants which appear to be predominantly in either an outwardly or an inwardly facing conformation. When the latter two mutations are combined, sugar-dependent IIA(Glc) binding returns to near wild-type levels. The findings suggest that binding of various substrates to lactose permease results in a collection of unique conformations, each of which presents a specific surface toward the inner face of the membrane that can interact to varying degrees with IIA(Glc).     

Processivity, substrate binding, and mechanism of cellulose hydrolysis by Thermobifida fusca Cel9A

Thermobifida fusca Cel9A-90 is a processive endoglucanase consisting of a family 9 catalytic domain (CD), a family 3c cellulose binding module (CBM3c), a fibronectin III-like domain, and a family 2 CBM. This enzyme has the highest activity of any individual T. fusca enzyme on crystalline substrates, particularly bacterial cellulose (BC). Mutations were introduced into the CD or the CBM3c of Cel9A-68 using site-directed mutagenesis. The mutant enzymes were expressed in Escherichia coli; purified; and tested for activity on four substrates, ligand binding, and processivity. The results show that H125 and Y206 play an important role in activity by forming a hydrogen bonding network with the catalytic base, D58; another important supporting residue, D55; and Glc(-1) O1. R378, a residue interacting with Glc(+1), plays an important role in processivity. Several enzymes with mutations in the subsites Glc(-2) to Glc(-4) had less than 15% activity on BC and markedly reduced processivity. Mutant enzymes with severalfold-higher activity on carboxymethyl cellulose (CMC) were found in the subsites from Glc(-2) to Glc(-4). The CBM3c mutant enzymes, Y520A, R557A/E559A, and R563A, had decreased activity on BC but had wild-type or improved processivity. Mutation of D513, a conserved residue at the end of the CBM, increased activity on crystalline cellulose. Previous work showed that deletion of the CBM3c abolished crystalline activity and processivity. This study shows that it is residues in the catalytic cleft that control processivity while the CBM3c is important for loose binding of the enzyme to the crystalline cellulose substrate.     

Active site mapping of amylo-alpha-1,6-glucosidase in porcine liver glycogen debranching enzyme using fluorogenic 6-O-alpha-glucosyl-maltooligosaccharides

Glycogen debranching enzyme (GDE) has two enzymatic activities, 4-alpha-glucanotransferase and amylo-alpha-1,6-glucosidase. Products with 6-O-alpha-glucosyl structures formed from phosphorylase limit dextrin by the 4-alpha-glucanotransferase activity are hydrolyzed to glucose by the amylo-alpha-1,6-glucosidase activity. Here, we probed the active site of amylo-alpha-1,6-glucosidase in porcine liver GDE using various 6-O-alpha-glucosyl-pyridylamino (PA)-maltooligosaccharides, with structures (Glcalpha1-4)(m)(Glcalpha1-6)Glcalpha1-4(Glcalpha1-4)(n)GlcPA (GlcPA, 1-deoxy-1-[(2-pyridyl)amino]-D-glucitol residue). Fluorogenic dextrins were prepared from 6-O-alpha-glucosyl-alpha-, beta-, or gamma-cyclodextrin through partial acid hydrolysis, followed by fluorescent tagging of the reducing-end residues of the hydrolysates and separation by gel filtration and reversed-phase HPLC. Porcine liver GDE hydrolyzed dextrins with the structure Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glc to glucose and the corresponding PA-maltooligosaccharides, whereas other dextrins were not hydrolyzed. Thus, substrates must have two glucosyl residues sandwiching the isomaltosyl moiety to be hydrolyzed. The rate of hydrolysis increased as m increased and reached maximum at m = 4. The rates were the highest when n = 1 but did not vary much with changes in n. Of the dextrins examined, Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (6(3)-O-alpha-glucosyl-PA-maltoheptaose) was hydrolyzed most rapidly, suggesting that it fits the best in the amylo-alpha-1,6-glucosidase active site. It is likely that the active site accommodates 6(2)-O-alpha-glucosyl-maltohexaose and that the interactions of seven glucosyl residues with the active site allow the most rapid hydrolysis of the alpha-1,6-glucosidic linkage of the isomaltosyl moiety.     

Effect of cofactor binding and loop conformation on side chain methyl dynamics in dihydrofolate reductase

Dihydrofolate reductase (DHFR) has several flexible active site loops that facilitate ligand binding and catalysis. Previous studies of backbone dynamics in several complexes of DHFR indicate that the time scale and amplitude of motion depend on the conformation of the active site loops. In this study, information on dynamics is extended to methyl-containing side chains. To understand the role of side chain dynamics in ligand binding and loop conformation, methyl deuterium relaxation rates of Escherichia coli DHFR in binary folate and ternary folate:NADP+ complexes have been measured, together with chi(1) rotamer populations for threonine, isoleucine, and valine residues, determined from measurements of 3J(CgammaCO) and 3J(CgammaN) coupling constants. The results indicate that, in addition to backbone motional restriction in the adenosine-binding site, side chain flexibility in the active site and the surrounding active site loops is diminished upon binding NADP+. Resonances for several methyls in the active site and the surrounding active site loops were severely broadened in the folate:NADP+ ternary complex, suggesting the presence of motion on the chemical shift time scale. The side chains of Ile14 and Ile94, which pack against the nicotinamide and pterin rings of the cofactor and substrate, respectively, exhibit rotamer disorder in the ternary folate:NADP+ complex. Conformational fluctuations of these side chains may play a role in transition state stabilization; the observed line broadening for Ile14 suggests motions on a microsecond/millisecond time scale.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

Conformational flexibility of PEP mutase

Previous work has indicated that PEP mutase catalyzes the rearrangement of phosphoenolpyruvate to phosphonopyruvate by a dissociative mechanism. The crystal structure of the mutase with Mg(II) and sulfopyruvate (a phosphonopyruvate analogue) bound showed that the substrate is anchored to the active site by the Mg(II), and shielded from solvent by a large loop (residues 115-133). Here, the crystal structures of wild-type and D58A mutases, in the apo state and in complex with Mg(II), are reported. In both unbound and Mg(II)-bound states, the active site is accessible to the solvent. The loop (residues 115-133), which in the enzyme-inhibitor complexes covers the active site cavity, is partially disordered or adopts a conformation that allows access to the cavity. In the apo state, the residues associated with Mg(II) binding are poised to accept the metal ion. When Mg(II) binds, the coordination is the same as that previously observed in the enzyme-Mg(II) sulfopyruvate complex, except that the coordination positions occupied by two ligand oxygen atoms are occupied by two water molecules. When the loop opens, three key active site residues are displaced from the active site, Lys120, Asn122, and Leu124. Lys120 mediates Mg(II) coordination. Asn122 and Leu124 surround the transferring phosphoryl group, and thus prevent substrate hydrolysis. Amino acid replacement of any one of these three loop residues results in a significant loss of catalytic activity. It is hypothesized that the loop serves to gate the mutase active site, interconverting between an open conformation that allows substrate binding and product release and a closed conformation that separates the reaction site from the solvent during catalysis.     

Switched or not?: the structure of unphosphorylated CheY bound to the N terminus of FliM

Phosphorylation of Escherichia coli CheY increases its affinity for its target, FliM, 20-fold. The interaction between BeF(3)(-)-CheY, a phosphorylated CheY (CheY approximately P) analog, and the FliM sequence that it binds has been described previously in molecular detail. Although the conformation that unphosphorylated CheY adopts in complex with FliM was unknown, some evidence suggested that it is similar to that of CheY approximately P. To resolve the issue, we have solved the crystallographic structure of unphosphorylated, magnesium(II)-bound CheY in complex with a synthetic peptide corresponding to the target region of FliM (the 16 N-terminal residues of FliM [FliM(16)]). While the peptide conformation and binding site are similar to those of the BeF(3)(-)-CheY-FliM(16) complex, the inactive CheY conformation is largely retained in the unphosphorylated Mg(2+)-CheY-FliM(16) complex. Communication between the target binding site and the phosphorylation site, observed previously in biochemical experiments, is enabled by a network of conserved side chain interactions that partially mimic those observed in BeF(3)(-)-activated CheY. This structure makes clear the active role that the beta4-alpha4 loop plays in the Tyr(87)-Tyr(106) coupling mechanism that enables allosteric communication between the phosphorylation site and the target binding surface. Additionally, this structure provides a high-resolution view of an intermediate conformation of a response regulator protein, which had been generally assumed to be two state.     

The solution NMR structure of glucosylated N-glycans involved in the early stages of glycoprotein biosynthesis and folding.

Glucosylated oligomannose N-linked oligosaccharides (Glc(x)Man9GlcNAc2 where x = 1-3) are not normally found on mature glycoproteins but are involved in the early stages of glycoprotein biosynthesis and folding as (i) recognition elements during protein N-glycosylation and chaperone recognition and (ii) substrates in the initial steps of N-glycan processing. By inhibiting the first steps of glycan processing in CHO cells using the alpha-glucosidase inhibitor N-butyl-deoxynojirimycin, we have produced sufficient Glc3Man7GlcNAc2 for structural analysis by nuclear magnetic resonance (NMR) spectroscopy. Our results show the glucosyl cap to have a single, well-defined conformation independent of the rest of the saccharide. Comparison with the conformation of Man9GlcNAc2, previously determined by NMR and molecular dynamics, shows the mannose residues to be largely unaffected by the presence of the glucosyl cap. Sequential enzymatic cleavage of the glucose residues does not affect the conformation of the remaining saccharide. Modelling of the Glc3Man9GlcNAc2, Glc2Man9GlcNAc2 and Glc1Man9GlcNAc2 conformations shows the glucose residues to be fully accessible for recognition. A more detailed analysis of the conformations allows potential recognition epitopes on the glycans to be identified and can form the basis for understanding the specificity of the glucosidases and chaperones (such as calnexin) that recognize these glycans, with implications for their mechanisms of action.     

Engineering the exo-loop of Trichoderma reesei cellobiohydrolase, Cel7A. A comparison with Phanerochaete chrysosporium Cel7D

The exo-loop of Trichoderma reesei cellobiohydrolase Cel7A forms the roof of the active site tunnel at the catalytic centre. Mutants were designed to study the role of this loop in crystalline cellulose degradation. A hydrogen bond to substrate made by a tyrosine at the tip of the loop was removed by the Y247F mutation. The mobility of the loop was reduced by introducing a new disulphide bridge in the mutant D241C/D249C. The tip of the loop was deleted in mutant Delta(G245-Y252). No major structural disturbances were observed in the mutant enzymes, nor was the thermostability of the enzyme affected by the mutations.The Y247F mutation caused a slight k(cat) reduction on 4-nitrophenyl lactoside, but only a small effect on cellulose hydrolysis. Deletion of the tip of the loop increased both k(cat) and K(M) and gave reduced product inhibition. Increased activity was observed on amorphous cellulose, while only half the original activity remained on crystalline cellulose. Stabilisation of the exo-loop by the disulphide bridge enhanced the activity on both amorphous and crystalline cellulose. The ratio Glc(2)/(Glc(3)+Glc(1)) released from cellulose, which is indicative of processive action, was highest with Tr Cel7A wild-type enzyme and smallest with the deletion mutant on both substrates. Based on these data it seems that the exo-loop of Tr Cel7A has evolved to facilitate processive crystalline cellulose degradation, which does not require significant conformational changes of this loop.     

Interplay of catalytic subsite residues in the positioning of α-d-glucose 1-phosphate in sucrose phosphorylase

Kinetic and molecular docking studies were performed to characterize the binding of α-d-glucose 1-phosphate (αGlc 1-P) at the catalytic subsite of a family GH-13 sucrose phosphorylase (from L. mesenteroides) in wild-type and mutated form. The best-fit binding mode of αGlc 1-P dianion had the phosphate group placed anti relative to the glucosyl moiety (adopting a relaxed ⁴C₁ chair conformation) and was stabilized mainly by hydrogen bonds from residues of the enzyme?s catalytic triad (Asp¹⁹⁶, Glu²³⁷ and Asp²⁹⁵) and from Arg¹³⁷. Additional feature of the αGlc 1-P docking pose was an intramolecular hydrogen bond (2.7 Å) between the glucosyl C2-hydroxyl and the phosphate oxygen. An inactive phosphonate analog of αGlc 1-P did not show binding to sucrose phosphorylase in different experimental assays (saturation transfer difference NMR, steady-state reversible inhibition), consistent with evidence from molecular docking study that also suggested a completely different and strongly disfavored binding mode of the analog as compared to αGlc 1-P. Molecular docking results also support kinetic data in showing that mutation of Phe⁵², a key residue at the catalytic subsite involved in transition state stabilization, had little effect on the ground-state binding of αGlc 1-P by the phosphorylase. However, when combined with a second mutation involving one of the catalytic triad residues, the mutation of Phe⁵² by Ala caused complete (F52A_D196A; F52A_E237A) or very large (F52A_D295A) disruption of the proposed productive binding mode of αGlc 1-P with consequent effects on the enzyme activity. Effects of positioning of αGlc 1-P for efficient glucosyl transfer from phosphate to the catalytic nucleophile of the enzyme (Asp¹⁹⁶) are suggested. High similarity between the αGlc 1-P conformers bound to sucrose phosphorylase (modeled) and the structurally and mechanistically unrelated maltodextrin phosphorylase (experimental) is revealed.     

Crystal structures of Melanocarpus albomyces cellobiohydrolase Cel7B in complex with cello-oligomers show high flexibility in the substrate binding

Cellobiohydrolase from Melanocarpus albomyces (Cel7B) is a thermostable, single-module, cellulose-degrading enzyme. It has relatively low catalytic activity under normal temperatures, which allows structural studies of the binding of unmodified substrates to the native enzyme. In this study, we have determined the crystal structure of native Ma Cel7B free and in complex with three different cello-oligomers: cellobiose (Glc₂), cellotriose (Glc₃), and cellotetraose (Glc₄), at high resolution (1.6–2.1 Å). In each case, four molecules were found in the asymmetric unit, which provided 12 different complex structures. The overall fold of the enzyme is characteristic of a glycoside hydrolase family 7 cellobiohydrolase, where the loops extending from the core β-sandwich structure form a long tunnel composed of multiple subsites for the binding of the glycosyl units of a cellulose chain. The catalytic residues at the reducing end of the tunnel are conserved, and the mechanism is expected to be retaining similarly to the other family 7 members. The oligosaccharides in different complex structures occupied different subsite sets, which partly overlapped and ranged from −5 to +2. In four cellotriose and one cellotetraose complex structures, the cello-oligosaccharide also spanned over the cleavage site (−1/+1). There were surprisingly large variations in the amino acid side chain conformations and in the positions of glycosyl units in the different cello-oligomer complexes, particularly at subsites near the catalytic site. However, in each complex structure, all glycosyl residues were in the chair (⁴C₁) conformation. Implications in relation to the complex structures with respect to the reaction mechanism are discussed.     

Role of magnesium ions in DNA recognition by the EcoRV restriction endonuclease

The restriction endonuclease EcoRV binds two magnesium ions. One of these ions, Mg(A)(2+), binds to the phosphate group where the cleavage occurs and is required for catalysis, but the role of the other ion, Mg(B)(2+) is debated. Here, multiple independent molecular dynamics simulations suggest that Mg(B)(2+) is crucial for achieving a tightly bound protein-DNA complex and stabilizing a conformation that allows cleavage. In the absence of Mg(B)(2+) in all simulations the protein-DNA hydrogen bond network is significantly disrupted and the sharp kink at the central base pair step of the DNA, which is observed in the two-metal complex, is not present. Also, the active site residues rearrange in such a way that the formation of a nucleophile, required for DNA hydrolysis, is unlikely.