Fig. 3

Structure/function analysis of beneficial mutations in GH3 BGLs. a Molecular mapping of mutations using the A. aculeatus BGL1 crystal structure (PDB 4IIB). Chain A orange; Chain B gray. Substitutions identified by mutagenesis and functional selection are shown in magenta and labeled on Chain A. b Residues contributing to the substrate-binding pocket of A. aculeatus BGL1. c Gly²⁹⁴–Gly³¹³ residues (magenta) coordinate Phe³⁰⁵ (green) in the +1 subsite. d Alignments of GH3 residues. Residues identified by directed evolution are underlined. Asterisks (*) indicate beneficial substitutions found in nature. A. acu Aspergillus aculeatus BGL1; A. nig Aspergillus niger BGL1; A. nid Apergillus nidulans; A. fum Aspergillus fumigatus; N. cra Neurospora crassa; F. gra Fusarium graminearum; P. bla Phycomyces blaskesleeanus; U. may Ustilago maydis; C. cin Coprinopsis cinerea; R. ory Rhizopus oryzae. Color coding corresponds between (b), (c), and (d). Multiple sequence alignments were performed using Clustal Omega [73]. Structural analyses were performed using PyMOL