Improving the alkalophilic performances of the Xyll xylanase from Streptomyces sp S38:: Structural comparison and mutational analysis

Endo-beta-1,4-xylanases of the family 11 glycosyl-hydrolases are catalytically active over a wide range of pH. Xyll from Streptomyces sp. S38 belongs to this family, and its optimum pH for enzymatic activity is 6. Xynll from Bacillus agaradhaerens and Xylj from Bacillus sp. 4 1 M-I share 85% sequence identity and have been described as highly alkalophilic enzymes. In an attempt to better understand the alkalophilic adaptation of xylanases, the three-dimensional structures of Xynll and Xyll were compared. This comparison highlighted an increased number of salt-bridges and the presence of more charged residues in the catalytic cleft as well as an eight-residue-longer loop in the alkalophilic xylanase Xynll. Some of these charges were introduced in the structure of Xyl I by site-directed mutagenesis with substitutions Y16D, S18E, G50R,N92D, A135Q.EI39K, and Y186E. Furthermore, the eight additional loop residues of Xynll were introduced in the homologous loop of Xyll. In addition, the coding sequence of the XylJ catalytic domain was synthesized by recursive PCR, expressed in a Streptomyces host, purified, and characterized together with the Xyll mutants. The Y186E substitution inactivated Xyl I, but the activity was restored when this mutation was combined with the G50R or S18E substitutions. Interestingly, the E139K mutation raised the optimum pH of Xyl I from 6 to 7.5 but had no effect when combined with the N92D substitution. Modeling, studies identified the possible formation of an interaction between the introduced lysine and the substrate, which could be eliminated by the formation of a putative salt-bridge in the N92D/E139K mutant.