Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH

Xylanase C from Aspergillus kawachii has an optimum pH of 2.0 and is stable at pH 1.0, The crystal structure of xylanase C was determined at 2.0 Angst rom resolution (R-factor 19.4%). The overall structure was similar to those of other family 11 xylanases. Asp37 and an acid-base catalyst, Glu170, are located at a hydrogen-bonding distance (2.8 Angstrom), as in other xylanas es with low pH optima. Asp37 of xylanase C was replaced with asparagine and other residues by site-directed mutagenesis, Analyses of the wild-type and mutant enzymes showed that Asp37 is important for high enzyme activity at low pH, In the case of the asparagine mutant, the optimum pH shifted to 5.0 and the maximum specific activity decreased to about 15% of that of the wi ld-type enzyme, On structural comparison with xylanases with higher pH opti ma, another striking feature of the xylanase C structure was found; the enz yme has numerous acidic residues concentrated on the surface (so-called 'Se r/Thr surface' in most family 11 xylanases). The relationship of the stabil ity against extreme pH conditions and high salt concentrations with the spa cially biased distribution of charged residues on the proteins is discussed .