REFINED CRYSTAL-STRUCTURE OF THE CATALYTIC DOMAIN OF XYLANASE-A FROM PSEUDOMONAS-FLUORESCENS AT 1.8 ANGSTROM RESOLUTION

The three-dimensional structure of native xylanase A from Pseudomonas fluorescens subspecies cellulosa has been refined at 1.8 Angstrom reso lution. The space group is P2(1)2(1)2(1) with four molecules in the as ymmetric unit. The final model has an R factor of 0.166 for 103 749 re flections with the four molecules refined independently. The tertiary structure consists of an eightfold beta/alpha-barrel, the so-called TI M-barrel fold. The active site is in an open cleft at the carboxy-term inal 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 th ese catalytic glutamate residues are found on beta-bulges. An atypical ly long loop after strand 7 is stabilized by calcium. Unusual features include a non-proline cis-peptide residue Ala80 which is found on a b eta-bulge at the end of beta-strand 3. The three beta-bulge type disto rtions occurring on beta-strands 3, 4 and 7 are functionally significa nt as they serve to orient important active-site residues. The active- site residues are further held in place by an extensive hydrogen-bondi ng network of active-site residues in the catalytic site of xylanase A . A chain of well ordered water molecules occupies the substrate-bindi ng cleft, some or all of which are expelled on binding of the substrat e.