The structure of a family 110 glycoside hydrolase provides insight into the hydrolysis of α-1,3-galactosidic linkages in λ-carrageenan and blood group antigens

Abstract

α-Linked galactose is a common carbohydrate motif in nature that is processed by a variety of glycoside hydrolases from different families. Terminal Galα1-3Gal motifs are found as a defining feature of different blood group and tissue antigens, as well as the building block of the marine algal galactan λ-carrageenan. The blood group B antigen and linear α-Gal epitope can be processed by glycoside hydrolases in family GH110, whereas the presence of genes encoding GH110 enzymes in polysaccharide utilization loci from marine bacteria suggests a role in processing λ-carrageenan. However, the structure-function relationships underpinning the α-1,3-galactosidase activity within family GH110 remain unknown. Here we focus on a GH110 enzyme (PdGH110B) from the carrageenolytic marine bacterium Pseudoalteromonas distincta U2A. We showed that the enzyme was active on Galα1-3Gal but not the blood group B antigen. X-ray crystal structures in complex with galactose and unhydrolyzed Galα1-3Gal revealed the parallel β-helix fold of the enzyme and the structural basis of its inverting catalytic mechanism. Moreover, an examination of the active site reveals likely adaptations that allow accommodation of fucose in blood group B active GH110 enzymes or, in the case of PdGH110, accommodation of the sulfate groups found on λ-carrageenan. Overall, this work provides insight into the first member of a predominantly marine clade of GH110 enzymes while also illuminating the structural basis of α-1,3-galactoside processing by the family as a whole.

Keywords: Pseudoalteromonas; X-ray crystal structure; X-ray crystallography; blood group antigen; carrageenan; enzyme structure; galactose; galactosidase; glycoside hydrolase; structural biology.

Figure 1

Activity of PdGH110B on α-1,3– and β-1,4–galactobiose.A, galactose release curve showing release of galactose units by PdGH110B when incubated with αG2 (closed squares) and βG2 (closed triangles), along with a d-galactose standard (closed circles). B, kinetic analysis of PdGH110B activity on αG2 at 25 °C using a galactose release kit to measure units of galactose released. In both panels, error bars show standard deviations of measurements made in triplicate.

Figure 2

Structural features of PdGH110B in complex with D-galactose.A, the structure of the PdGH110B dimer. Monomer A (purple) is shown in cartoon representation, and monomer B (gray) is shown as a solvent-accessible surface representation. B, cartoon representation showing the different structural domains of PdGH110B, the β-helix domain (purple), domain I (gray), and domain II (dark gray). C, the association of the domain II α-helix extending into the neighboring chain's active site. The coloring is the same as in A. D, the active site pocket, shown as a solvent-accessible surface in gray, sequesters the galactose residue. The O1, which would be engaged in a glycosidic linkage, is indicated. E, the interaction of the galactose residue with the likely catalytic acid residue. Interacting side chains are shown in purple, and the water molecule is shown as a red sphere. In all A–E, the D-galactose monosaccharide is represented as yellow sticks.

Figure 3

PdGH110B D344N in complex with αG2.A, the specific interactions of αG2 with the PdGH110B active site. Residues forming the −1 and +1 subsites are represented as purple sticks, and the catalytic machinery is shown as transparent pink sticks. αG2 is represented as yellow sticks, water molecules are red spheres, and hydrogen bonds are shown as dashed lines. B, the solvent-accessible surface of the PdGH110B dimer shown as electrostatic potential. Positive and negative surface potential are shown in blue and red, respectively. The O₂ that may bear a sulfate residue in λ-carrageenan is indicated. The dashed line defines the interface between the two monomers of the PdGH11B dimer. In both panels the active site subsites and sugar residues are indicated in green.

Figure 4

PdGH110B using an inverting catalytic mechanism.A, ¹H NMR spectra of pNP-α-d-galactopyranoside treated with PdGH110B measured at various time points. Signals with NMR chemical shifts corresponding with those distinctive of pNP-α-d-galactopyranoside, α-galactopyranoside, and β-galactopyranoside are labeled with integrated peak areas shown where applicable. B, αG2 (yellow sticks) bound in the active site of PdGH110B D344N. The three catalytic residues are represented as purple sticks, hydrogen bonds are dashed lines, and the water molecule is a red sphere. C, an overlay of the PdGH110B αG2 complex (purple sticks) with the GH49 isopullulanase from Aspergillus niger (green; PDB code 2z8g) (34), the GH28 exo-polygalacturonase from Yersinia enterocolitica (yellow; PDB code 2uvf) (36), and the GH87 α-1,3-glucanase from Paenibacillus glycanilyticus (gray; PDB code 6k0n) (35). The active site subsites are labeled in green. The putative acid is indicated with A, and the pair of putative bases is indicated with B.

Figure 5

Phylogeny and conservation of glycoside hydrolase family 110 sequences.A, phylogenetic tree of family GH110 constructed from 334 sequences extracted from the CAZy database. The numbered arms are as follows: arm 1, BtGal110A from Bacteroides thetaiotaomicron; arm 2, BtGal110B from B. thetaiotaomicron; arm 3, BfGalA from Bacteroides fragilis; arm 4, SaGal110A from Streptomyces avermitilis; arm 5, BbAgaBb from Bifidobacterium bifidum; and arm 6, BfGal110B from B. fragilis. The shaded region indicates the “marine” clade. See also Fig. S4 and Table S1 for complete annotation of the tree. B, the conservation of active site residues of GH110 mapped onto the structure of PdGH110B by ConSurf analysis (46). C, conservation of the active site shown as accessible surface representation. The color scheme representing degree of residue conservation in B and C is shown in B. Active site subsites and sugar residues are labeled in green.