Conservation in the mechanism of glucuronoxylan hydrolysis revealed by the structure of glucuronoxylan xylanohydrolase (CtXyn30A) fromClostridium thermocellum

Abstract

Glucuronoxylan endo-β-1,4-xylanases cleave the xylan chain specifically at sites containing 4-O-methylglucuronic acid substitutions. These enzymes have recently received considerable attention owing to their importance in the cooperative hydrolysis of heteropolysaccharides. However, little is known about the hydrolysis of glucuronoxylans in extreme environments. Here, the structure of a thermostable family 30 glucuronoxylan endo-β-1,4-xylanase (CtXyn30A) from Clostridium thermocellum is reported. CtXyn30A is part of the cellulosome, a highly elaborate multi-enzyme complex secreted by the bacterium to efficiently deconstruct plant cell-wall carbohydrates. CtXyn30A preferably hydrolyses glucuronoxylans and displays maximum activity at pH 6.0 and 70°C. The structure of CtXyn30A displays a (β/α) TIM-barrel core with a side-associated β-sheet domain. Structural analysis of the CtXyn30A mutant E225A, solved in the presence of xylotetraose, revealed xylotetraose-cleavage oligosaccharides partially occupying subsites -3 to +2. The sugar ring at the +1 subsite is held in place by hydrophobic stacking interactions between Tyr139 and Tyr200 and hydrogen bonds to the OH group of Tyr227. Although family 30 glycoside hydrolases are retaining enzymes, the xylopyranosyl ring at the -1 subsite of CtXyn30A-E225A appears in the α-anomeric configuration. A set of residues were found to be strictly conserved in glucuronoxylan endo-β-1,4-xylanases and constitute the molecular determinants of the restricted specificity displayed by these enzymes. CtXyn30A is the first thermostable glucuronoxylan endo-β-1,4-xylanase described to date. This work reveals that substrate recognition by both thermophilic and mesophilic glucuronoxylan endo-β-1,4-xylanases is modulated by a conserved set of residues.

A New Subfamily of Glycoside Hydrolase Family 30 with Strict Xylobiohydrolase Function

“…Otherwise, a single xylose remains yielding XGX. These results and further deductions agree with known GH30 protein structure and xylan binding interactions concerning the orientation of xylose positioned in the +1 subsite (Freire et al, 2016). No other products were detected from these reactions.…”

RETRACTED: Thermostable xylanases from thermophilic fungi and bacteria: Current perspective

“…The distance between these carboxyl residues is less than 5Ǻ thus support retention mechanism for hydrolysis. CtXyn30A is active over a broad range of pH and displays an optimum temperature of 70 o C. The thermostability of the enzyme may be attributed to the presence of higher number of salt bridges and strong hydrogen bonding between the side chain residues (Freire et al, 2016).…”

A plasmid borne, functionally novel glycoside hydrolase family 30 subfamily 8 endoxylanase from solventogenic Clostridium

“…With the GlcA of the XGX positioned as in Ec XynA, numerous clashes would occur with the β8–α8 loop region of Ca Xyn30A. Comparison of the positive subsite region of Ca Xyn30A ( B ) superposed with the newly available xylobiose ligand-bound structure model of Ct Xyn30A (PDB code: 5A6M) [ 50 ] provides an approximation of how xylobiose may coordinate into the positive subsite region. Not previously known is the hydrogen-bonding role that Tyr232 may have in ligand coordination on the +1 subsite.…”

“…A recent publication of the ligand-bound structure of the canonical GH30-8 endoxylanase Xyn30A from Clostridium thermocellum ( Ct Xyn30A) [ 50 ] has revealed how xylobiose interacts within the positive subsite region of a Gram-positive form of GH30-8 enzymes ( Figure 9B ) [ 51 ]. Generally, all GH30-8 enzymes appear to benefit from a similar positive subsite region hydrophobic cleft as formed in Ca Xyn30A by Trp144 on the N-terminal face and by Tyr209 on the C-terminal face, with Leu181 forming the cleft floor.…”