Identification of a structural mechanism for prediction and discrimination of substrates for multifunctional GH5...

Multifunctional and heat-resistant enzymes with both cellulose and hemicellulose degrading activities are particularly useful in converting plant biomass into biofuel. We previously characterised a bifunctional cellulase/xylanase CtCel5E from Clostridium thermocellum and a bifunctional cellulase/mannanase TmCel5A from Thermotoga maritima. In this study, we further characterised another GH5 enzyme, CtCel5T, annotated as a trifunctional cellulase/xylanase/mannanase. Despite the highly conserved amino acid sequences and crystal structures, they exhibit distinct hemicellulase substrate specificities. Through structural comparison coupled with site-directed mutagenesis, we identified that Met277 in loop 6 and Glu360 in loop 8 of CtCel5T overlapped with His205 and Trp210 in loop 6 of TmCel5A, respectively, but no amino acid at these spatial positions in CtCel5E, suggesting that these residues may play critical roles in discriminating hemicellulose substrates. In agreement with this, exchanging the amino acids in these loops of CtCel5T and TmCel5A switched substrate preference. Moreover, using phylogenetic tree analyses to confirm the GH5 family of cellulases/hemicellulases with functions consistent with these structural features, we could predict the functions of relatively uncharacterised enzymes using MtGlu5, a GH5 endoglucanase from Meiothermus taiwanensis WR-220, as an example. This study thus identified a structural mechanism for determining the substrate specificities of GH5 multifunctional cellulases/hemicellulases.