Crystal structure at 1.8 angstrom resolution and proposed amino acid sequence of a thermostable xylanase from Thermoascus aurantiacus

Thermoascus aurantiacus xylanase is a thermostable enzyme which hydrolyses xylan, a major hemicellulose component in the biosphere. Crystals belonging to P2(1) space group with a = 41.7 Angstrom, b = 68.1 Angstrom c = 51.4 An gstrom and beta = 113.6 degrees, Z = 2 were grown that could diffract to be tter than 1.8 Angstrom resolution. The structure was solved by molecular re placement method using the Streptomyces lividans xylanase model. The amino acid sequence was determined from the electron density map aided by multipl e alignment of related xylanase sequences. The sequence thus obtained provi des a correction to the sequence reported earlier based on biochemical meth ods. The final refined protein model at 1.8 Angstrom resolution with 301 am ino acid residues and 266 water molecules has an R-factor of 16.0 % and fre e R of 21.1% with good stereochemistry. The single polypeptide chain assume s (alpha/beta)(8) TIM-barrel fold and belongs to F/10 family of glycoside h ydrolases. The active site consists of two glutamate residues located at th e C terminus end of the beta-barrel, conforming to the double displacement mechanism for the enzyme action. A disulphide bond and more than ten salt b ridges have been identified. In particular, the salt bridge Arg124-Glu232 w hich is almost buried, bridges the beta-strands beta 4 and beta 7 where the catalytic glutamate residues reside, and it may play a key role in the sta bility and activity at elevated temperature. To our knowledge, for the firs t time in the F/10 family xylanases, we observe a proline residue in the mi ddle of the alpha-helix alpha 6 which may be contributing to better packing . Earlier studies show that the enzyme retains its activity even at 70 degr ees C. The refined protein model has allowed a detailed comparison with the other known structures in the F/10 family of enzymes. The possible causati ve factors for thermostability are discussed. (C) 1999 Academic Press.