Site-directed mutagenesis study of a conserved residue in family 10 glycanases: histidine 86 of xylanase A from Streptomyces lividans |
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Authors: | Roberge M; Shareck F; Morosoli R; Kluepfel D; Dupont C |
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Affiliation: | Centre de Recherche en Microbiologie Appliquee, Institut Armand- Frappier, Universite du Quebec, Laval, Canada. |
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Abstract: | 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)/dGdnxHCl] 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.
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