Structure of a PL17 family alginate lyase demonstrates functional similarities among exotype depolymerases

Abstract

Brown macroalgae represent an ideal source for complex polysaccharides that can be utilized as precursors for cellulosic biofuels. The lack of recalcitrant lignin components in macroalgae polysaccharide reserves provides a facile route for depolymerization of constituent polysaccharides into simple monosaccharides. The most abundant sugars in macroalgae are alginate, mannitol, and glucan, and although several classes of enzymes that can catabolize the latter two have been characterized, studies of alginate-depolymerizing enzymes have lagged. Here, we present several crystal structures of Alg17c from marine bacterium Saccharophagus degradans along with structure-function characterization of active site residues that are suggested to be involved in the exolytic mechanism of alginate depolymerization. This represents the first structural and biochemical characterization of a family 17 polysaccharide lyase enzyme. Despite the lack of appreciable sequence conservation, the structure and β-elimination mechanism for glycolytic bond cleavage by Alg17c are similar to those observed for family 15 polysaccharide lyases and other lyases. This work illuminates the evolutionary relationships among enzymes within this unexplored class of polysaccharide lyases and reinforces the notion of a structure-based hierarchy in the classification of these enzymes.

Keywords: Algae; Alginate Lyase; Biofuel; Carbohydrate Processing; Enzyme Catalysis; Protein Structure.

FIGURE 1.

Chemical structures of various uronic acid-containing polymers found in alginate and products and polymer specificity of Alg17c. A, Haworth projections showing polymers of G, M, and heteropolymers sequences of poly(MG). Exolytic depolymerization of these polysaccharides by alginate lyase yields a monosaccharide and a product containing a Δ-(4,5)-unsaturated uronic acid moiety. B, thin layer chromatographic analysis of polymer specificity of Alg17c. Polysaccharide standards shown are for monomer (Lane 1, d-glucose) and dimer (Lane 2, d-trehalose). A mixture of alginate di-, tri-, and tetrasaccharides (Lane 3) are processed into mono- and disaccharides (Lane 4) in the presence of Alg17c. DP, degree of polymerization.

FIGURE 2.

Crystal structure of the PL17 family Alg17c from S. degradans 2-40. A, overall structure of the Alg17c monomer showing the α₆/α₆-barrel (pink) and antiparallel β-sheet (green) domains. The zinc ion is colored in red. B, structure of the Alg17c biological dimer. C, structure-based multiple sequence alignments of characterized PL17 family enzymes, including Alg17c, AlgL (from Sphingomonas sp. MJ3), and AlyIII (from Pseudomonas sp. OS-ALG9). The first 24 amino acids encode the signal sequence and are enclosed in the green rectangle. Secondary structural elements are derived from the Alg17c structure, and residues involved in substrate binding (green triangles), reaction chemistry (red diamonds), and metal ion binding (blue circles) are indicated as described.

FIGURE 3.

Conservation of structure among enzymatically diverse polysaccharide lyases. A comparison of the crystal structures of Alg17c (colored in pink and green) (A) with Atu3025 from A. tumefaciens (in blue and brown; Protein Data Bank code 3A0O) (B), heparinase II form P. heparinus (in umber and lavender with the zinc ion in red; Protein Data Bank code 2FUQ) (C), and hyaluronate lyase from Streptococcus pneumonia (in cyan and tan; Protein Data Bank code 1LXK) (D) is shown.

FIGURE 4.

Difference Fourier maps of the bound metal ion. A stereoview of the Alg17c metal-binding site derived from the refined coordinates of Alg17c is shown. The Alg17c carbon atoms are shown in yellow ball-and-stick representation, metal-bound solvent molecules are colored in black, and the metal is shown in red. Superimposed is a difference Fourier electron density map (contoured at 5σ over background in blue and 15.0σ in red) calculated with coefficients |F_obs| − |F_calc| and phases from the final refined model with the coordinates of the metal deleted prior to one round of refinement. Based on the coordination chemistry and the metal assignment in hyaluronate lyase, the metal is putatively assigned as a zinc ion.

FIGURE 5.

Isothermal titration calorimetric analysis. Binding isotherms characterizing the interaction between a ΔMMG trisaccharide with the Y258A variant of Alg17c are shown. Data were fit to a single site model. deg, degrees.

FIGURE 6.

Alg17c active site with bound trisaccharide. A, stereoview of the Alg17c active site derived from the co-crystal structure in complex with ΔMMG. The Alg17c carbon atoms are shown in yellow ball-and-stick representation, and the trisaccharide is shown in green. Superimposed is a difference Fourier electron density map (contoured at 2.7σ over background in blue and 6.0σ in red) calculated with coefficients |F_obs| − |F_calc| and phases from the final refined model with the coordinates of the trisaccharide deleted prior to one round of refinement. B, comparison of the overall structure of Alg17c in the absence (pink) and presence (cyan) of bound oligosaccharide substrate. C, close-up view of the Alg17c active site in the absence (pink) and presence (cyan) of substrate.

FIGURE 7.

Mechanism of alginate depolymerization by Alg17c. Depolymerization of urinate-containing polysaccharides is carried out using the Asn²⁰¹ and His²⁰² pair to stabilize the negative charge at the carboxylate, Tyr⁴⁵⁰ as a general base to abstract the proton at C5, and Tyr²⁵⁸ to serve as a general acid to donate a proton to the oxygen of the glycosidic bond.