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Structural Basis for Xyloglucan Specificity and α‑d‑Xyl<i>p</i>(1 → 6)‑d‑Glc<i>p</i> Recognition at the −1 Subsite within the GH5 Family
GH5
is one of the largest glycoside hydrolase families, comprising at
least 20 distinct activities within a common structural scaffold.
However, the molecular basis for the functional differentiation among
GH5 members is still not fully understood, principally for xyloglucan
specificity. In this work, we elucidated the crystal structures of
two novel GH5 xyloglucanases (XEGs) retrieved from a rumen microflora
metagenomic library, in the native state and in complex with xyloglucan-derived
oligosaccharides. These results provided insights into the structural
determinants that differentiate GH5 XEGs from parental cellulases
and a new mode of action within the GH5 family related to structural
adaptations in the −1 subsite. The oligosaccharide found in
the XEG5A complex, permitted the mapping, for the first time, of the
positive subsites of a GH5 XEG, revealing the importance of the pocket-like
topology of the +1 subsite in conferring the ability of some GH5 enzymes
to attack xyloglucan. Complementarily, the XEG5B complex covered the
negative subsites, completing the subsite mapping of GH5 XEGs at high
resolution. Interestingly, XEG5B is, to date, the only GH5 member
able to cleave XXXG into XX and XG, and in the light of these results,
we propose that a modification in the −1 subsite enables the
accommodation of a xylosyl side chain at this position. The stereochemical
compatibility of the −1 subsite with a xylosyl moiety was also
reported for other structurally nonrelated XEGs belonging to the GH74
family, indicating it to be an essential attribute for this mode of
action