tailieunhanh - Báo cáo khoa học: Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases

The secondary substrate binding site (SBS) ofBacillus subtilisandAspergil-lus niger glycoside hydrolase family 11 xylanases was studied by site-direc-ted mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. | IFEBS Journal Secondary substrate binding strongly affects activity and binding affinity of Bacillus subtilis and Aspergillus niger GH11 xylanases Sven Cuyvers Emmie Dornez Mohammad N. Rezaei Annick Pollet Jan A. Delcour and Christophe M. Courtin Laboratory of Food Chemistry and Biochemistry Leuven Food Science and Nutrition Research Centre LFoRCe Katholieke Universiteit Leuven Belgium Keywords arabinoxylan GH11 noncatalytic binding single domain xylanase surface binding Correspondence C. Courtin Laboratory of Food Chemistry and Biochemistry Leuven Food Science and Nutrition Research Centre LFoRCe Katholieke Universiteit Leuven Kasteelpark Arenberg 20 - PO Box 2463 B-3001 Leuven Belgium Fax 32 16 321997 Tel 32 16 321917 E-mail Received 12 October 2010 revised 11 January 2011 accepted 20 January 2011 doi The secondary substrate binding site SBS of Bacillus subtilis and Aspergillus niger glycoside hydrolase family 11 xylanases was studied by site-directed mutagenesis and evaluation of activity and binding properties of mutant enzymes on different substrates. Modification of the SBS resulted in an up to three-fold decrease in the relative activity of the enzymes on polymeric versus oligomeric substrates and highlighted the importance of several amino acids in the SBS forming hydrogen bonds or hydrophobic stacking interactions with substrates. Weakening of the SBS increased Kd values by up to 70-fold in binding affinity tests using natural substrates. The impact that modifications in the SBS have both on activity and on binding affinity towards polymeric substrates clearly shows that such structural elements can increase the efficiency of these single domain enzymes on their natural substrates. Introduction Glycoside hydrolases can possess noncatalytic polysaccharide binding sites that facilitate attack on the natural substrate. Most of these sites belong to separate domains referred to as .