Insight into the role of substrate-binding residues in conferring substrate specificity for the multifunctional polysaccharide lyase Smlt1473

Abstract

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 the 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 (poly-GlcUA), and poly-β-d-mannuronic acid (poly-ManA) in a pH-regulated manner. Here, we utilize a sequence structure guided approach based on a homology model of Smlt1473 to identify nine 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 toward poly-ManA and poly-GlcUA, respectively. Furthermore, a W171A mutant nearly eliminated HA activity, while increasing poly-ManA and poly-GlcUA activity by at least 35%. The effect of these mutations was analyzed by comparison with the high resolution structure of Sphingomonas sp. A1-III alginate lyase in complex with poly-ManA tetrasaccharide and by taking into account the structural differences between HA, poly-GlcUA, and poly-ManA. Overall, our results demonstrate that even minor changes in active site architecture have a significant effect on the substrate specificity of Smlt1473, whose structural plasticity could be applied to the design of highly active and specific polysaccharide lyases.

Keywords: Carbohydrate Processing; Enzyme Mechanism; Enzyme Mutation; Lyase; Polysaccharide; Protein Design; Substrate Specificity.

FIGURE 1.

Identification of putative substrate-binding residues in Smlt1473. A, surface homology model of Smlt1473 constructed with Swiss-Model and Protein Data Bank file 1QAZ as a template. A deep trench-like cleft was identified (black arrow, inset). Catalytically active residues are highlighted in white. Positively charged residues are highlighted in black. A positive patch was identified at the entrance of the active site, immediately adjacent to the catalytically active residues. B, sequence alignment of Smlt1473 with Sphingomonas sp. A1-III lyase (PDB code 1QAZ) (11). Identical residues are highlighted in gray. Residues predicted to be located in the active site cleft and participate in substrate binding are highlighted in black. Residues predicted to participate in the catalytic mechanism of Smlt1473 are marked by asterisks. Residues located in positive patch are marked by daggers.

FIGURE 2.

Specific activity of wild-type and mutant Smlt1473 against HA (A), poly-GlcUA (B), and poly-ManA (C). Purified wild-type and mutant Smlt1473 (14 μg) was mixed with 1 mg/ml HA in 30 mm sodium acetate, pH 5, or poly-ManA in 30 mm Tris, pH 9, in a total reaction volume of 350 μl. For poly-GlcUA, the amount of enzyme added to 1 mg/ml poly-GlcUA in 30 mm Tris, pH 7, was reduced to 1.4 μg due to higher activity. Enzymatic activity was monitored by absorbance at 235 nm. One unit of activity was defined as an increase in absorbance at 235 nm of 1.0 per min at 25 °C. Dashed lines indicate wild-type activity for comparison with other mutants. All reactions were performed in triplicate, and error is reported as standard deviation.

FIGURE 3.

Location of putative substrate-binding residues in Smlt1473. A–D, homology model of Smlt1473 built with Swiss-Model and Protein Data Bank code 1QAZ. Images were generated in PyMOL. Predicted catalytic residues (Asn¹⁶⁷, His¹⁶⁸, Arg²¹⁵, and Tyr²²²) are highlighted in white. Putative substrate-binding residues of interest are highlighted in black. E–H, percent change in specific activity of mutant lyases from wild-type Smlt1473 for HA at pH 5, poly-GlcUA at pH 7, and poly-ManA at pH 9. Enzymatic activity was monitored by change in absorbance at 235 nm. All reactions were performed in triplicate, and error is reported as standard deviation. Residues are organized into four groups as follows: residues located in the positive patch (Lys⁴², Lys¹⁶², Arg¹⁶³, and Arg²¹⁸; A and E) and residues whose mutation favors poly-ManA cleavage (His²²¹, Tyr²²⁵; B and F), poly-GlcUA cleavage (Tyr¹¹⁵, Arg³¹²; C and G), and poly-ManA/poly-GlcUA cleavage (Trp¹⁷¹; D and H).

FIGURE 4.

Percent change in enzyme efficiency (k_cat/K_m) of Smlt1473 mutants from wild type for poly-GlcUA at pH 7 and poly-ManA at pH 9.

FIGURE 5.

Heat map of putative substrate-binding residues with respect to substrate specificity. Surface model (A) and stick model (B) of Smlt1473 built with Swiss-Model and Protein Data Bank code 1QAZ. Images were generated in PyMOL. Predicted catalytic residues (Asn¹⁶⁷, His¹⁶⁸, Arg²¹⁵, and Tyr²²²) are highlighted in white. Each putative substrate-binding residue is colored according to the effect of mutating each residue on activity toward HA (red), poly-GlcUA (green), and poly-ManA (blue). The specific activity of the mutants against each substrate was scaled to the intensity of the corresponding color. The hydrogen bond between Tyr²²⁵ and Arg³¹² is highlighted by a yellow dashed line.

FIGURE 6.

Structural differences between poly-ManA and poly-GlcUA. Top, chair diagram of GlcUA and ManA, which are C2 epimers of each other. The C2 hydroxyl group is pointing down in the equatorial position for GlcUA and pointing up in the axial position for ManA. Bottom, three-dimensional structure of poly-GlcUA in a 2₁ helix (41) and poly-ManA in 3₂ helix (42). Structures were downloaded from PolySac3Db and rendered in PyMOL.