Refined crystal structure of the catalytic domain of xylanase A from Pseudomonas fluorescens at 1.8 A resolution

Abstract

The three-dimensional structure of native xylanase A from Pseudomonas flouorescens subspecies cellulosa has been refined at 1.8 A resolution. The space group is P2(1)2(1)2(1) 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 beta/alpha-barrel, the so-called TIM-barrel fold. The active site is in an open cleft at the carboxy-terminal end of the beta/alpha-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 beta-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 beta-bulge at the end of beta-strand 3. The three beta-bulge type distortions occurring on beta-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.