Insight into the role of substrate binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473 [Microbiology]
May 13th, 2014 by MacDonald, L. C., Berger, B. W.
Anionic polysaccharides are of growing interest in the biotechnology industry due to their potential pharmaceutical applications in drug delivery and wound treatment. Chemical composition and polymer length strongly influence the physical and biological properties of the polysaccharide and thus its potential industrial and medical applications. One promising approach to determining monomer composition and controlling degree of polymerization involves the use of polysaccharide lyases, which catalyze the depolymerization of anionic polysaccharides via a β-elimination mechanism. Utilization of these enzymes for the production of custom-made oligosaccharides requires a high degree of control over substrate specificity. Previously we characterized a polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia k279a which exhibited significant activity against hyaluronan (HA), poly-β-D-glucuronic acid (polyGlcA), and poly-β-D-mannuronic acid (polyManA) in a pH-regulated manner. Here we utilize a sequence-structure guided approach based on a homology model of Smlt1473 to identify 9 putative substrate-binding residues and examine their effect on substrate specificity via site-directed mutagenesis. Interestingly, single point mutations H221F and R312L resulted in increased activity and specificity towards polyManA and polyGlcA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing polyManA and polyGlcA activity by at least 35%. The effect of these mutations were analyzed by comparison with the high-resolution structure of Spingomonas sp. A1-III alginate lyase in complex with polyManA tetrasaccharide and by taking into account the structural differences between HA, polyGlcA, and polyManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, and similar approaches can be used to select highly active and specific polysaccharide lyases.