SITE-DIRECTED MUTAGENESIS STUDY OF A CONSERVED RESIDUE IN FAMILY-10 GLYCANASES - HISTIDINE-86 OF XYLANASE-A FROM STREPTOMYCES-LIVIDANS

Xylanases from family 10 glycanases contain three conserved histidine residues in their active site. The role of H86 in the structure-functi on of xylanase A from Streptomyces lividans (XlnA) was studied by site -directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produ ced, purified and characterized. The six mutations reduced the affinit y of XlnA towards xylan without having any major effect on the catalyt ic constant. All these mutations also lowered the pK(a) of the acid-ba se catalyst by 0.46-1.94 pH units. The mutations decreased the enzyme stability at 60 degrees C by up to 95% and the transition temperature by 2.2-5.8 degrees C. Unfolding of the protein with guanidine hydrochl oride (Gdn.HCl) showed that five out of six mutations decreased the co ncentration required to denature 50% of the XlnA, confirming the impor tance of H86 for the stability of the enzyme. The increase in m value {m = d(Delta G)/d[Gdn.HCl]} also suggested the involvement of residue H86 in the structure of the denatured state of XlnA, It can be conclud ed from this study that this active site residue was conserved in fami ly 10 glycanases for its function in maintaining the elevated pK(a) of the acid-base catalyst and in the stability of the protein, while bei ng of little importance for the activity.