Aromatic residues surrounding the active site tunnel of TfCel48A influence activity, processivity, and synergistic interactions with other cellulases

Cel48A of Thermobifida fusca (TfCel48A) is a processive exocellulase that contains an active site tunnel and digests lignocellulosic biomass via synergistic interactions between different cellulases. Cel48A possesses a number of aromatic amino acids lining the tunnel entrance, which are highly conserved across a diverse number of microbial species and appear to play a role in the selection and threading of individual strands of cellulose from highly recalcitrant substrates. In this study, we sought to further elucidate the roles of these tunnel entrance aromatic amino acids by creating a series of double mutants and examining their effect on TfCel48A activity, processivity, and synergistic interactions with the well‐studied processive endocellulase TfCel9A. Our results provide further insight concerning the mechanism of Cel48A kinetics with soluble and insoluble substrates and could play an influential role in the application of Cel48A and other exocellulases for industrial purposes. TfCel48A containing cellohexaose as substrate, longitudinal view. Location of aromatic amino acids residues described in this paper are illustrated. Tryptophan residues described previously by M. Chen et al. (2016) (W424, W148, W315 and W313) as substrate binding are indicated in blue. Y97 and Y213 are shown in light green. F195 is shown in orange. Cellohexaose substrate and cellobiose product are depicted in red. Inset depicts a time course digestion profile of TfCel48A on BC (blue) and PASC (red).