The tertiary structure at 1.59 angstrom resolution and the proposed amino acid sequence of a family-11 xylanase from the thermophilic fungus Paecilomyces varioti Bainier

We report the crystal structure at 1.59 Angstrom and the proposed amino aci d sequence of are endo-1,4-beta-xylanase (PVX) from the thermophilic fungus Paecilomyces varioti Bainier (PvB), stable up to 75 degrees C. This fungus is attracting clinical attention as a pathogen causing post-surgical infec tions. Its xylanase, known as a skin-contact allergen, is the first protein from this fungus whose three-dimensional structure has been elucidated. Th e crystals of PVX conform to the space group P2(1)2(1)2(1) with a = 38.76 A ngstrom, b = 54.06 Angstrom and c = 90.06 Angstrom. The structure was solve d by molecular replacement techniques using polyalanine coordinates of the Thermomyces lanuginosus xylanase (PDB code 1YNA) and a careful model buildi ng based on the amino acid sequence known for two trypsin-digested peptide fragments (17 residues), the sequence and structural alignment of family-11 xylanases and electron density maps. The final refined model has 194 amino acid residues and 128 water molecules, with a crystallographic R-factor of 19.07% and a free R-factor of 21.94%. The structure belongs to an all-beta fold, with two curved beta-sheets, forming the cylindrical active-site cle ft, and a lone alpha-helix, as present in other family-11 xylanases. We hav e carried out a quantitative comparison of the structure and sequence of th e present thermophilic xylanase (PVX) with other available native structure s of mesophiles and thermophiles, the first such detailed analysis to be ca rried out on family-11 xylanases. The analysis provides a basis for the rat ionalisation of the idea that the "hinge" region is made more compact in th ermophiles by the addition of a disulphide bridge between Cys110 and Cys154 and a N-H ... O hydrogen bond between Trp159 near the extremity of the lon e alpha-helix and Trp138 on beta-strand B8. This work brings out explicitly the presence of the C-H ... O and the C-H ...pi type interactions in these enzymes. A complete description of structural stability of these enzymes n eeds to take account of these weaker interactions. (C) 2000 Academic Press.