Geotrichum sp. M128 possesses two xyloglucan-specific
glycoside hydrolases belonging to family 74,
xyloglucan-specific endo-beta-1,4-glucanase (XEG) and oligoxyloglucan reducing-end-specific
cellobiohydrolase (OXG-RCBH). Despite their similar
amino acid sequences (48% identity), their modes of action and
substrate specificities are distinct. XEG
catalyzes the hydrolysis of
xyloglucan polysaccharides in endo mode, while OXG-RCBH acts on
xyloglucan oligosaccharides at the
reducing end in exo mode. Here, we determined the
crystal structure of XEG at 2.5 A resolution, and compared it to a previously determined structure of OXG-RCBH. For the most part, the
amino acid residues that interact with
substrate are conserved between the two
enzymes. However, there are notable differences at subsite positions -1 and +2. OXG-RCBH has a loop around the +2 site that blocks one end of the
active site cleft, which accounts for its exo mode of action. In contrast, XEG lacks a corresponding loop at this site, thereby allowing binding to the middle of the main chain of the
substrate. At the -1 site in OXG-RCBH, Asn488 interacts with the
xylose side chain of the
substrate, whereas the -1 site is occupied by Tyr457 in XEG. To confirm the contribution of this residue to
substrate specificity, Tyr457 was substituted by
Gly in XEG. The
wild-type XEG cleaved the oligoxyloglucan at a specific site; the Y457G variant cleaved the same
substrate, but at various sites. Together, the absence of a loop in the
cleft and the presence of bulky Tyr457 determine the
substrate specificity of XEG.
