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-function of xylanase A from Streptomyces lividans (XlnA) was studied by site-directed mutagenesis. Six mutant proteins (H86A/E/F/K/Q/W) were produced, purified and characterized. The six mutations reduced the affinity of XlnA towards xylan without having any major effect on the catalytic constant. All these mutations also lowered the pKa of the acid-base 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 hydrochloride (GdnxHCl) showed that five out of six mutations decreased the concentration required to denature 50% of the XlnA, confirming the importance of H86 for the stability of the enzyme. The increase in m value ¿m=d(deltaG)/d[GdnxHCl]¿ also suggested the involvement of residue H86 in the structure of the denatured state of XlnA. It can be concluded from this study that this active site residue was conserved in family 10 glycanases for its function in maintaining the elevated pKa of the acid-base catalyst and in the stability of the protein, while being of little importance for the activity.
Centre de Recherche en Microbiologie Appliquée, Institut Armand-Frappier, Université du Québec, Laval, Canada.
M
M
Roberge
F
F
Shareck
R
R
Morosoli
D
D
Kluepfel
C
C
Dupont
D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D002384:Catalysis; D002942:Circular Dichroism; D043364:Endo-1,4-beta Xylanases; D006639:Histidine / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D013816:Thermodynamics; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014990:Xylans; D006639:Histidine; D014995:Xylosidases; D043364:Endo-1,4-beta Xylanases