Search

Glycoside hydrolase family 7 (GH7) cellulases are key enzymes responsible for carbon cycling on earth through their role in cellulose degradation and constitute highly important industrial enzymes as well. Although these enzymes are found in a wide variety of evolutionarily distant organisms across eukaryotes, they exhibit remarkably conserved features within two groups: exo-acting cellobiohydrolases and endoglucanases. However, recently reports have emerged of a separate clade of GH7 endoglucanases from protist symbionts of termites that are 60-80 amino acids shorter. In this work, we describe the first crystal structure of a short GH7 endoglucanase, RsSymEG1, from a symbiont of the lower termite Reticulitermes speratus. A more open flat surface and shorter loops around the non-reducing end of the cellulose-binding cleft indicate enhanced access to cellulose chains on the surface of cellulose microfibrils. Additionally, when comparing activities on polysaccharides to a typical fungal GH7 endoglucanase (Trichoderma longibrachiatum Cel7B), RsSymEG1 showed significantly faster initial hydrolytic activity. We also examine the prevalence and diversity of GH7 enzymes that the symbionts provide to the termite host, compare overall structures and substrate binding between cellobiohydrolase and long and short endoglucanase, and highlight the presence of similar short GH7s in other organisms., (© 2023 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Glycoside hydrolase family 7 (GH7) cellobiohydrolases (CBHs) play a key role in biomass recycling in nature. They are typically the most abundant enzymes expressed by potent cellulolytic fungi, and are also responsible for the majority of hydrolytic potential in enzyme cocktails for industrial processing of plant biomass. The thermostability of the enzyme is an important parameter for industrial utilization. In this study, Cel7 enzymes from different fungi were expressed in a fungal host and assayed for thermostability, including Hypocrea jecorina Cel7A as a reference. The most stable of the homologues, Humicola grisea var. thermoidea Cel7A, exhibits a 10°C higher melting temperature (T(m) of 72.5°C) and showed a 4-5 times higher initial hydrolysis rate than H. jecorina Cel7A on phosphoric acid-swollen cellulose and showed the best performance of the tested enzymes on pretreated corn stover at elevated temperature (65°C, 24 h). The enzyme shares 57% sequence identity with H. jecorina Cel7A and consists of a GH7 catalytic module connected by a linker to a C-terminal CBM1 carbohydrate-binding module. The crystal structure of the H. grisea var. thermoidea Cel7A catalytic module (1.8 Å resolution; R(work) and R(free) of 0.16 and 0.21, respectively) is similar to those of other GH7 CBHs. The deviations of several loops along the cellulose-binding path between the two molecules in the asymmetric unit indicate higher flexibility than in the less thermostable H. jecorina Cel7A.

The filamentous fungus Hypocrea jecorina (anamorph of Trichoderma reesei) is the predominant source of enzymes for industrial saccharification of lignocellulose biomass. The major enzyme, cellobiohydrolase Cel7A, constitutes nearly half of the total protein in the secretome. The performance of such enzymes is susceptible to inhibition by compounds liberated by physico-chemical pre-treatment if the biomass is kept unwashed. Xylan and xylo-oligosaccharides ( XOS) have been proposed to play a key role in inhibition of cellobiohydrolases of glycoside hydrolase family 7. To elucidate the mechanism behind this inhibition at a molecular level, we used X-ray crystallography to determine structures of H. jecorina Cel7A in complex with XOS. Structures with xylotriose, xylotetraose and xylopentaose revealed a predominant binding mode at the entrance of the substrate-binding tunnel of the enzyme, in which each xylose residue is shifted ~ 2.4 Å towards the catalytic center compared with binding of cello-oligosaccharides. Furthermore, partial occupancy of two consecutive xylose residues at subsites -2 and -1 suggests an alternative binding mode for XOS in the vicinity of the catalytic center. Interestingly, the -1 xylosyl unit exhibits an open aldehyde conformation in one of the structures and a ring-closed pyranoside in another complex. Complementary inhibition studies with p-nitrophenyl lactoside as substrate indicate mixed inhibition rather than pure competitive inhibition. Database The atomic coordinates and structure factors are available in the Protein Data Bank under accession number ( H. jecorina Cel7A E212Q variant, complex with xylotriose), ( H. jecorina Cel7A E217Q variant, complex with xylotriose), ( H. jecorina Cel7A E212Q variant, complex with xylopentaose), ( H. jecorina Cel7A E217Q variant, complex with xylopentaose), (wild-type H. jecorina Cel7A, complex with xylopentaose) and ( H. jecorina Cel7A E217Q variant, complex with xylotetraose). [ABSTRACT FROM AUTHOR]

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 article discusses a research on fungal cellulases. Topics include structures, microfibrils, and substrates of cellulose, glycoside hydrolase catalytic mechanisms, and early developments in fungal cellulases. Other topics include carbohydrate-binding modules and linkers, structural studies and catalytic function of glycoside hydrolases, and kinetic modeling and visualization.

Cellobiohydrolases (CBHs) are typically major components of natural enzyme cocktails for biomass degradation. Their active sites are enclosed in a tunnel, enabling processive hydrolysis of cellulose chains. Glycoside hydrolase Family 6 (GH6) CBHs act from nonreducing ends by an inverting mechanism and are present in many cellulolytic fungi and bacteria. The bacterial Thermobifida fusca Cel6B (TfuCel6B) exhibits a longer and more enclosed active site tunnel than its fungal counterparts. Here, we determine the structures of two TfuCel6B mutants co-crystallized with cellobiose, D274A (catalytic acid), and the double mutant D226A/S232A, which targets the putative catalytic base and a conserved serine that binds the nucleophilic water. The ligand binding and the structure of the active site are retained when compared with the wild type structure, supporting the hypothesis that these residues are directly involved in catalysis. One structure exhibits crystallographic waters that enable construction of a model of the α-anomer product after hydrolysis. Interestingly, the product sites of TfuCel6B are completely enclosed by an "exit loop" not present in fungal GH6 CBHs and by an extended "bottom loop". From the structures, we hypothesize that either of the loops enclosing the product subsites in the TfuCel6B active site tunnel must open substantially for product release. With simulation, we demonstrate that both loops can readily open to allow product release with equal probability in solution or when the enzyme is engaged on cellulose. Overall, this study reveals new structural details of GH6 CBHs likely important for functional differences among enzymes from this important family. [ABSTRACT FROM AUTHOR]