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1H NMR spectroscopy has been used to analyze the product profiles arising from the hydrolysis of cellooligosaccharides by family GH9 cellulases. The product profiles obtained with the wild type and several active site mutants of a bacterial processive endoglucanase, TfCel9A, were compared with those obtained by a randomly acting plant endoglucanase, PttCel9A. PttCel9A is an orthologue of the Arabidopsis endocellulase, Korrigan, which is required for efficient cellulose biosynthesis. As expected, poplar PttCel9A was shown to catalyze the degradation of cellooligosaccharides by inversion of the configuration of the anomeric carbon. The product analyses showed that the number of interactions between the glucose units of the substrate and the aromatic residues in the enzyme active sites determines the point of cleavage in both enzymes.

Trichoderma reesei cellobiohydrolase Cel6A is an inverting glycosidase. Structural studies have established that the tunnel-shaped active site of Cel6A contains two aspartic acids, D221 and D175, that are close to the glycosidic oxygen of the scissile bond and at hydrogen-bonding distance from each other. Here, site-directed mutagenesis, X-ray crystallography, and enzyme kinetic studies have been used to confirm the role of residue D221 as the catalytic acid. D175 is shown to affect protonation of D221 and to contribute to the electrostatic stabilization of the partial positive charge in the transition state. Structural and modeling studies suggest that the single-displacement mechanism of Cel6A may not directly involve a catalytic base. The value of (D2O)(V) of 1.16 +/- 0.14 for hydrolysis of cellotriose suggests that the large direct effect expected for proton transfer from the nucleophilic water through a water chain (Grotthus mechanism) is offset by an inverse effect arising from reversibly breaking the short, tight hydrogen bond between D221 and D175 before catalysis.

Methylumbelliferyl-β-cellobioside ( MUF- G2) is a convenient fluorogenic substrate for certain β-glycoside hydrolases ( GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes ( GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF- G2 in the +1 subsite of Hypocrea jecorina Cel6 A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-β-cellobiosides [6-chloro-4-methyl- (Cl MUF); 6-chloro-4-trifluoromethyl- (ClF3 MUF); 4-phenyl- (Ph UF); 6-chloro-4-phenyl- (ClPh UF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6 A was 10-150 times higher than with MUF- G2, although it was still three orders of magnitude lower than with H. jecorina Cel7 B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while Cl MUF- G2 could be used for determination of kcat and KM for H. jecorina Cel6 A and Thermobifida fusca Cel6 A. Crystal structures of H. jecorina Cel6 A D221 A mutant soaked with the MUF-, Cl MUF- and ClPh UF-β-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF- G2. Database Structural data have been submitted to the Protein Data Bank under accession numbers pdb 4AU0, 4AX7, 4AX6 Structured digital abstract • http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7260296 • Cel6A and Cel6A bind by x-ray crystallography (View Interaction: 1, 2) [ABSTRACT FROM AUTHOR]

The cellulase mixture of Hypocrea jecorina (formerly Trichoderma reesei) contains a variety of exo- and endoglucanases that belong to different structural families. As such, these enzymes form an interesting model system to study the enzyme-ligand interactions in glycoside hydrolases. The nucleophilic carboxylate of retaining β-glycosidases is believed to form a hydrogen bond with the 2-hydroxyl group of their substrate. Consequently, replacing this hydroxyl group with an amino group should result in a stronger electrostatic interaction and thus an increased affinity for the ligand. In this study, several modified cellobiosides were synthesized and evaluated as cellulase inhibitors. The introduction of an amino group was found to have an unpredictable effect on the inhibitory power of the ligands. However, the enzymes display a very high affinity for the corresponding 2-azido compounds, precursors in the synthetic route. The new ligand m-iodobenzyl 2-deoxy-2-azido-β-cellobioside even is the strongest inhibitor of cellobiohydrolase I known to date (KI = 1 μM). [ABSTRACT FROM AUTHOR]