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

Substrate binding sites of SCXyl.

<p>A) Molecular surface of chain A, with the Trp281-Arg291 loop from chain B and CmXyn10B shown as green and blue lines, respectively. B) Stereo view of the glycone region (from −3 to −1 subsites) of SCXyl (carbon atoms in green) and CmXyn10B (carbon atoms in blue). C) Stereo view of the aglycone region (from +1 to +4 subsites). The substrate found in the CmXyn10B-complex structure (PDB code: 1UQY) is shown in Figs. A, B and C, as ball-and-sticks. Residues numbering refers to the SCXyl enzyme.</p

SCXy1 analysis by SAXS.

<p>(A) Experimental SAXS curve of the SCXyl and fitting procedures, and experimental distance distribution function. An inset containing the corresponding Guinier plot. (B) DR model (aminoacids are represented by cyan dummy residues and salvation shell by gray dummy residues) superposed with SCXyl crystallographic monomer structure).</p

Biophysical characterization of SCXy11.

<p><i>(</i>A) Far UV-CD spectrum of SCXyl at 20°C and (B) Thermal stability of SCXyl by CD.</p

Phylogenetic relationships among members of Glycoside Hydrolase Family 10.

<p>The phylogenetic tree was generated using the amino acid sequences from bacterial, fungal and uncultured microorganisms GH10 family members and one bacterial GH11 member (root). This tree was constructed using MEGA 4.0 software by the Neighbour Joining (NJ) method. The Bootstrap values (n = 1000 replicates) are indicated as percentage at the node of phylogenetic tree.</p

Temperature and pH profile of SCXyl.

<p>(A) The surface response and (B) contour curves generated in the central composite rotatable design (CCRD) illustrate the effect of the pH and temperature on the xylanase activity.</p

SAXS structural parameters of endoxylanase SCXy1.

<p>SAXS structural parameters of endoxylanase SCXy1.</p

Analysis of the breakdown products released by SCXyl.

<p>Xylotriose, xylotetraose, xylopentaose and xylohexaose degradation profiles are represented, respectively, (A), (B), (C) and (D). The intermediary products after 30 min of incubation are represented. The detached small boxes show the final degradation products, which were always xylose and xylobiose. The black and white arrows depict the preferential and the less preferential SCXyl cleavage site, respectively, based on the profile of intermediary products formed.</p

The three-dimensional structure of SCXyl.

<p>A) Cartoon representation, colored by secondary structure, with the Trp281-Arg291 loop (chain B) colored in green and the catalytic residues shown as sticks. B) Surface electrostatic potential, colored from negative (red) to positive (blue) charge. The ordered loop in chain B is shown as a green line. C) Superposition of CmXyn10B (PDB code 1UQY) on SCXyl structure, with divergent regions in blue and green, respectively. The substrate from the CmXyn10B-complex structure is represented as ball-and-sticks with carbon atoms in yellow.</p

The effect of SCXyl treatment on PASB prior the saccharification with commercial cellulase preparation.

<p>The pre-treatment step with SCXyl followed by addition of ACCELLERASE® 1500 (cellulolytic enzymatic cocktail) improved PASB saccharification.</p