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.
Highlights
The ABH glycan antigens define the ABO blood types, the appropriate matching of which is a key consideration in blood transfusions and organ transplantations
This pair of proteins displayed 98 and 94% amino acid sequence identity to two putative orthologous GH110 enzymes in P. carrageenovora 9T that are encoded by adjacent genes in a presumed l-carrageenan polysaccharide utilization locus (PUL) [5]
The GH110 family was initially identified by examination of members that removed the immunodominant a-1,3–linked galactose residues of blood group B antigen
Summary
U2A was isolated from the marine environment for its capacity to grow on macroalgal polysaccharides, including carrageenan, as previously described [6]. A structural homology search using the DALI server [22] identified a fold most similar to those of a GH87 a-1,3-glucanase from Bacillus circulans [23], as well as two epimerases, AlgE4 and AlgE6, from Azotobacter vinelandii [16] (PDB code 5LW3) This core b-helix is surrounded by two small b-barrel domains (domains I and II) that contribute to the residues involved in dimerization of PdGH110B (Fig. 2B). GH110 enzymes were previously shown to operate through use of a single displacement, or inverting, catalytic mechanism [1] We confirmed this for PdGH110B by using 1H NMR to monitor the initial release of the b-anomer of D-galactose from pNP-a-D-galactopyranoside, which indicates inversion of the anomeric configuration of C1 involved in the glycosidic bond (Fig. 4A). A water molecule that sits 3.5 Å beneath C1 of the D-galactose residue in the 21 subsite is suitably positioned to be activated as a nucleophile by Asp-321 and/or Asp-345 (Fig. 4B)
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