Refined Crystal Structure of the Catalytic Domain of Xylanase A from Pseudomonas fluorescens at 1.8 Å Resolution

The three-dimensional structure of native xylanase A from Pseudomonas flouorescens subspecies cellulosa has been refined at 1.8 Å resolution. The space group is P2₁2₁2₁ with four molecules in the asymmetric unit. The final model has an R factor of 0.166 for 103 749 reflections with the four molecules refined independently. The tertiary structure consists of an eightfold β/α-barrel, the so-called TIM-barrel fold. The active site is in an open cleft at the carboxy-terminal end of the β/α-barrel, and the active-site residues are a pair of glutamates, Glu127 on strand 4 and Glu246 on strand 7. Both these catalytic glutamate residues are found on β-bulges. An atypically long loop after strand 7 is stabilized by calcium. Unusual features include a non-proline cis-peptide residue Ala80 which is found on a β-bulge at the end of β-strand 3. The three β-bulge type distortions occurring on β-strands 3, 4 and 7 are functionally significant as they serve to orient important active-site residues. The active-site residues are further held in place by an extensive hydrogen-bonding network of active-site residues in the catalytic site of xylanase A. A chain of well ordered water molecules occupies the substrate-binding cleft, some or all of which are expelled on binding of the substrate.