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
2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês
10.1074/jbc.ra120.015776
ISSN1083-351X
AutoresBailey E. McGuire, A.G. Hettle, Chelsea Vickers, Dustin T. King, David J. Vocadlo, A.B. Boraston,
Tópico(s)Carbohydrate Chemistry and Synthesis
Resumoα-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. 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. The ABH glycan antigens define the ABO blood types, the appropriate matching of which is a key consideration in blood transfusions and organ transplantations. The O-blood group, defined by the smaller H antigen, is considered a universal donor. Accordingly, enzymatic conversion of the more elaborate and immunogenic A/B antigens to the H antigen provides an attractive route to avoid antigen mismatching and create a ready supply of universal donor blood. A campaign to identify enzymes that could hydrolyze terminal α-1,3–linked N-acetyl d-galactosamine and/or d-galactose from the A and B antigens, respectively, and thereby provide a set of biocatalytic tools to perform this antigen switching revealed a set of enzymes with specific B antigen exo-α-1,3-galactosidase activity (1Liu Q.P. Yuan H. Bennett E.P. Levery S.B. Nudelman E. Spence J. Pietz G. Saunders K. White T. Olsson M.L. Henrissat B. Sulzenbacher G. Clausen H. Identification of a GH110 subfamily of α1,3-galactosidases: novel enzymes for removal of the α3Gal xenotransplantation antigen.J. Biol. Chem. 2008; 283 (18227066): 8545-855410.1074/jbc.M709020200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Liu Q.P. Sulzenbacher G. Yuan H. Bennett E.P. Pietz G. Saunders K. Spence J. Nudelman E. Levery S.B. White T. Neveu J.M. Lane W.S. Bourne Y. Olsson M.L. Henrissat B. et al.Bacterial glycosidases for the production of universal red blood cells.Nat. Biotechnol. 2007; 25 (17401360): 454-46410.1038/nbt1298Crossref PubMed Scopus (200) Google Scholar). These enzymes were the founding members of GH110 (glycoside hydrolase 110) family, of which all currently characterized members are exo-α-1,3-galactosidases that are able to hydrolyze the B antigen glycan (Galα1–3(Fucα1–2)Gal-R) (1Liu Q.P. Yuan H. Bennett E.P. Levery S.B. Nudelman E. Spence J. Pietz G. Saunders K. White T. Olsson M.L. Henrissat B. Sulzenbacher G. Clausen H. Identification of a GH110 subfamily of α1,3-galactosidases: novel enzymes for removal of the α3Gal xenotransplantation antigen.J. Biol. Chem. 2008; 283 (18227066): 8545-855410.1074/jbc.M709020200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Liu Q.P. Sulzenbacher G. Yuan H. Bennett E.P. Pietz G. Saunders K. Spence J. Nudelman E. Levery S.B. White T. Neveu J.M. Lane W.S. Bourne Y. Olsson M.L. Henrissat B. et al.Bacterial glycosidases for the production of universal red blood cells.Nat. 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Most recently, genes encoding proteins that are classified by amino acid sequence identity into GH110 have been identified in polysaccharide utilization loci (PULs) from human gut microbiome bacteria and marine bacteria; these PULs are postulated to target λ-carrageenan, which is a polysaccharide found in marine algae (5Gobet A. Barbeyron T. Matard-Mann M. Magdelenat G. Vallenet D. Duchaud E. Michel G. Evolutionary evidence of algal polysaccharide degradation acquisition by Pseudoalteromonas carrageenovora 9T to adapt to macroalgal niches.Front. Microbiol. 2018; 9 (30524390)274010.3389/fmicb.2018.02740Crossref PubMed Scopus (35) Google Scholar, 6Hettle A.G. Hobbs J.K. Pluvinage B. Vickers C. Abe K.T. Salama-Alber O. McGuire B.E. Hehemann J.H. Hui J.P.M. Berrue F. Banskota A. Zhang J. Bottos E.M. Van Hamme J. Boraston A.B. Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.Commun. 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The backbone comprises d-galactose with alternating α-1,3- and β-1,4-linkages. The occurrence and specific patterns of sulfate esters on the free hydroxyl groups of the galactose backbone, along with the presence or absence of 3,6-anhydro-d-galactose, gives rise to numerous different carrageenan families (15Knutsen S.H. Myslabodski D.E. Larsen B. Usov A.I. A modified system of nomenclature for red algal galactans.Bot. Mar. 1994; 37: 163-17010.1515/botm.1994.37.2.163Crossref Scopus (375) Google Scholar, 16Usov A.I. Structural analysis of red seaweed galactans of agar and carrageenan groups.Food Hydrocoll. 1998; 12: 301-30810.1016/S0268-005X(98)00018-6Crossref Scopus (191) Google Scholar, 17Usov A. Polysaccharides of the red algae.Adv. Carbohydr. Chem. Biochem. 2011; 65 (21763512): 115-17910.1016/B978-0-12-385520-6.00004-2Crossref PubMed Scopus (206) Google Scholar). λ-Carrageenan is made of neocarrabiose motifs in which d-galactose-2,6-sulfate is α-1,3–linked to d-galactose-2-sulfate, and this disaccharide is joined by β-1,4-glycosidic linkages forming a linear λ-carrageenan polymer that is distinct from other carrageenans by its lack of 3,6-anhydro-d-galactose. Presently, how microbes process λ-carrageenan is poorly understood with only endo-acting β-1,4-λ-carrageenases from Pseudoalteromonas carrageenovora 9T (18Ohta Y. Hatada Y. A novel enzyme, λ-carrageenase, isolated from a deep-sea bacterium.J. Biochem. 2006; 140 (16926183): 475-48110.1093/jb/mvj180Crossref PubMed Scopus (39) Google Scholar, 19Guibet M. Colin S. Barbeyron T. Genicot S. Kloareg B. Michel G. Helbert W. Degradation of λ-carrageenan by Pseudoalteromonas carrageenovora λ-carrageenase: a new family of glycoside hydrolases unrelated to kappa- and iota-carrageenases.Biochem. J. 2007; 404 (17269933): 105-11410.1042/BJ20061359Crossref PubMed Scopus (73) Google Scholar) having been identified. To date, no other enzymes having activity consistent with λ-carrageenan processing have been experimentally identified, including the distinct absence of identified enzymes that are active on the α-1,3–linkages. We postulate that this is an activity performed by the GH110 enzymes found in λ-carrageenan PULs. Toward testing this hypothesis, we characterized the structure and function of PdGH110B. This enzyme is encoded by a gene we identified in the recently reported genome of Pseudoalteromonas distincta U2A (referred to as U2A for brevity) (6Hettle A.G. Hobbs J.K. Pluvinage B. Vickers C. Abe K.T. Salama-Alber O. McGuire B.E. Hehemann J.H. Hui J.P.M. Berrue F. Banskota A. Zhang J. Bottos E.M. Van Hamme J. Boraston A.B. Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.Commun. Biol. 2019; 2 (31886414): 47410.1038/s42003-019-0721-yCrossref PubMed Scopus (32) Google Scholar). PdGH110B has ∼25% amino acid sequence identity with the characterized GH110 enzymes from Bacteroides sp. (1Liu Q.P. Yuan H. Bennett E.P. Levery S.B. Nudelman E. Spence J. Pietz G. Saunders K. White T. Olsson M.L. Henrissat B. Sulzenbacher G. Clausen H. Identification of a GH110 subfamily of α1,3-galactosidases: novel enzymes for removal of the α3Gal xenotransplantation antigen.J. Biol. Chem. 2008; 283 (18227066): 8545-855410.1074/jbc.M709020200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 2Liu Q.P. Sulzenbacher G. Yuan H. Bennett E.P. Pietz G. Saunders K. Spence J. Nudelman E. Levery S.B. White T. Neveu J.M. Lane W.S. Bourne Y. Olsson M.L. Henrissat B. et al.Bacterial glycosidases for the production of universal red blood cells.Nat. Biotechnol. 2007; 25 (17401360): 454-46410.1038/nbt1298Crossref PubMed Scopus (200) Google Scholar) and ∼94% amino acid sequence identity with a putative GH110 enzyme present in the P. carrageenovora 9T PUL that is proposed to target λ-carrageenan (5Gobet A. Barbeyron T. Matard-Mann M. Magdelenat G. Vallenet D. Duchaud E. Michel G. Evolutionary evidence of algal polysaccharide degradation acquisition by Pseudoalteromonas carrageenovora 9T to adapt to macroalgal niches.Front. Microbiol. 2018; 9 (30524390)274010.3389/fmicb.2018.02740Crossref PubMed Scopus (35) Google Scholar). Here we demonstrate that PdGH110B is an α-galactosidase that can hydrolyze the Galα1–3Gal disaccharide, but not the blood group B-trisaccharide [Galα1–3(Fucα1–2)Gal], thus distinguishing it from previously characterized GH110 enzymes. The structural determination of PdGH110B in complex with enzyme substrate and products revealed the parallel β-helix fold of GH110 enzymes and a −1 subsite (20Davies G.J. Wilson K.S. Henrissat B. Nomenclature for sugar-binding subsites in glycosyl hydrolases.Biochem. J. 1997; 321 (9020895): 557-55910.1042/bj3210557Crossref PubMed Scopus (845) Google Scholar) that accommodates an unmodified galactose residue. An analysis of the structure points to a potential +1′ subsite that is key to accommodating sulfate modifications, as present in λ-carrageenan, or fucose, a defining constituent of the blood group B antigen glycan. Overall, the results are consistent with PdGH110B representing the founding member of a GH110 subfamily that exo-hydrolytically processes terminal α-linked galactose residues in λ-carrageenan while providing general insight into the specificity of the family as a whole. U2A was isolated from the marine environment for its capacity to grow on macroalgal polysaccharides, including carrageenan, as previously described (6Hettle A.G. Hobbs J.K. Pluvinage B. Vickers C. Abe K.T. Salama-Alber O. McGuire B.E. Hehemann J.H. Hui J.P.M. Berrue F. Banskota A. Zhang J. Bottos E.M. Van Hamme J. Boraston A.B. Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.Commun. Biol. 2019; 2 (31886414): 47410.1038/s42003-019-0721-yCrossref PubMed Scopus (32) Google Scholar). We identified two adjacent genes (locus tags EU511_08545 and EU511_08540) encoding proteins with 30% sequence identity to one another and ∼25% sequence identity to previously characterized GH110 enzymes. 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 λ-carrageenan PUL (5Gobet A. Barbeyron T. Matard-Mann M. Magdelenat G. Vallenet D. Duchaud E. Michel G. Evolutionary evidence of algal polysaccharide degradation acquisition by Pseudoalteromonas carrageenovora 9T to adapt to macroalgal niches.Front. Microbiol. 2018; 9 (30524390)274010.3389/fmicb.2018.02740Crossref PubMed Scopus (35) Google Scholar). A gene truncation of EU511_08540, encoding a protein we refer to as PdGH110B, lacking the predicted signal peptide was overproduced and purified, followed by qualitative assessment of activity on the synthetic substrates pNP-α-d-galactopyranoside and pNP-β-d-galactopyranoside. Recombinant PdGH110B showed activity only on pNP-α-d-galactopyranoside and a pH optimum of ∼5.6 (Fig. S1A). We further tested the activity of PdGH110B on more natural substrates using Galα1–3Gal (αG2) and Galβ1–4Gal (βG2), which represent the basic unmodified motifs present in λ-carrageenan, by quantifying galactose release. PdGH110B released d-galactose when incubated with αG2, whereas there was no activity on βG2 (Fig. 1A). The Km and kcat for αG2 were 5.9 ± 1.1 mm and 18.3 ± 0.002 s−1, respectively (Fig. 1B). Given the activity of other GH110 enzymes on the blood group B glycan, we tested PdGH110B using TLC but could not detect any activity (Fig. S1B). An initial preliminary structure of PdGH110B was determined by single-wavelength anomalous dispersion using a cadmium derivative. This initial model was used to solve the structure of PdGH110B in complex with a d-galactose monosaccharide to 2.35 Å resolution. The crystal structure of PdGH110B in complex with d-galactose revealed two chains in the asymmetric unit. Residues 25–238/241–616 for one molecule and residues 21–182/186–238/241–616 for the second molecule were modeled, with the missing residues residing in loop regions. The noncrystallographic dimer shows no evidence of being stable; however, each monomer in the asymmetric unit forms a crystallographic dimer (Fig. S2A). The crystallographic dimers (Fig. 2A) have a total molecular interface, determined by PISA (Proteins, Interfaces, Structures and Assemblies) (21Krissinel E. Henrick K. Inference of macromolecular assemblies from crystalline state.J. Mol. Biol. 2007; 372 (17681537): 774-79710.1016/j.jmb.2007.05.022Crossref PubMed Scopus (6801) Google Scholar) analysis, of 2300 Å2, predicting a stable dimeric state. Dynamic light scattering analysis of PdGH110B in solution at protein concentrations of 0.18, 0.37, and 0.73 mg/ml yielded a molecular mass of 136.3 ± 9.4 kDa. The expected molecular mass of the PdGH110B monomer is 67.9 kDa, resulting in an expected molecular mass of 135 kDa for a dimer. This indicates that PdGH110B adopts a dimeric quaternary structure, which is most likely the biologically relevant assembly. The overall fold of PdGH110B is that of a right-handed parallel β-helix of 11 complete turns. A structural homology search using the DALI server (22Holm L. DALI and the persistence of protein shape.Protein Sci. 2020; 29 (31606894): 128-14010.1002/pro.3749Crossref PubMed Scopus (324) Google Scholar) identified a fold most similar to those of a GH87 α-1,3-glucanase from Bacillus circulans (23Yano S. Suyotha W. Oguro N. Matsui T. Shiga S. Itoh T. Hibi T. Tanaka Y. Wakayama M. Makabe K. Crystal structure of the catalytic unit of GH 87-type α-1,3-glucanase Agl-KA from Bacillus circulans.Sci. Rep. 2019; 9 (31653959)1529510.1038/s41598-019-51822-5Crossref PubMed Scopus (6) Google Scholar), as well as two epimerases, AlgE4 and AlgE6, from Azotobacter vinelandii (16Usov A.I. Structural analysis of red seaweed galactans of agar and carrageenan groups.Food Hydrocoll. 1998; 12: 301-30810.1016/S0268-005X(98)00018-6Crossref Scopus (191) Google Scholar) (PDB code 5LW3). This core β-helix is surrounded by two small β-barrel domains (domains I and II) that contribute to the residues involved in dimerization of PdGH110B (Fig. 2B). The α-helix of domain II from the adjacent monomer folds over and along the wall of the active site, which was identified by a bound d-galactose, with several amino acid side chains protruding into the cleft that contains the active site pocket (Fig. 2C). The bound d-galactose monosaccharide was identified by clear electron density (Fig. S2B) found in the central region of the β-helix domain of both active sites of the dimer. The modeled monosaccharide occupied a pocket that sequesters the monosaccharide in a fashion that is typical for glycoside hydrolases that are exo-acting on the nonreducing end of glycans (Fig. 2D). Specifically, Asp-344 is located within hydrogen bonding distance of the C1-OH, where the scissile bond of an intact substrate would be (Fig. 2E), indicating that this residue is a likely candidate to play the catalytically essential role of general acid. To trap an intact substrate complex of the enzyme, we targeted this residue to generate an inactive D344N mutant, which indeed lacked activity on pNP-α-galactopyranoside. Crystals of the PdGH110B D344N mutant were soaked with an excess of αG2, and the structure was determined to 2.20 Å resolution. The refined structure revealed four monomers of PdGH110B D344N in the asymmetric unit. The monomers were organized as two noncrystallographic dimers with identical arrangements to the crystallographic dimer observed in the PdGH110B d-galactose complex, supporting the concept that the dimer is a stable quaternary structure. Clear electron density for the αG2 disaccharide was found in each monomer active site (Fig. S3) with the intact glycosidic linkage spanning the −1 subsite and +1 subsites and the catalytic machinery (Fig. 3A). There is an extended network of hydrogen bonds, as well as a single interacting aromatic residue, made between the αG2 molecule and the enzyme active site (Fig. 3A). The C2–C6 portion of the α-face of the d-galactose unit in the −1 subsite sits on a hydrophobic platform created by Trp-486, interacting through CH–π interactions often employed by CAZymes (24Spiwok V. CH/π interactions in carbohydrate recognition.Molecules. 2017; 22: 1012-103810.3390/molecules22071038Crossref Scopus (74) Google Scholar). The remainder of the −1 subsite is created by an extensive hydrogen bond network comprising interactions between Asn-85, Glu-488, Asn-348, and Glu-480 and the C3-OH, C4-OH, and C6-OH hydroxyl groups. Arg-265 coordinates both C2-OH and C3-OH, and Arg-453 interacts with C6-OH and the endocyclic oxygen (Fig. 3A). The +1 subsite is formed exclusively by the positively charged side chains of Arg-451, Arg-208, and through a water coordinated by Lys-207. The electrostatic potential of the active site surface indicates a generally acidic −1 subsite but a +1 subsite and neighboring surfaces that are basic (Fig. 3B). GH110 enzymes were previously shown to operate through use of a single displacement, or inverting, catalytic mechanism (1Liu Q.P. Yuan H. Bennett E.P. Levery S.B. Nudelman E. Spence J. Pietz G. Saunders K. White T. Olsson M.L. Henrissat B. Sulzenbacher G. Clausen H. Identification of a GH110 subfamily of α1,3-galactosidases: novel enzymes for removal of the α3Gal xenotransplantation antigen.J. Biol. Chem. 2008; 283 (18227066): 8545-855410.1074/jbc.M709020200Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We confirmed this for PdGH110B by using 1H NMR to monitor the initial release of the β-anomer of d-galactose from pNP-α-d-galactopyranoside, which indicates inversion of the anomeric configuration of C1 involved in the glycosidic bond (Fig. 4A). The architecture of the PdGH110B catalytic center is also consistent with an inverting catalytic mechanism (25Davies G. Henrissat B. Structures and mechanisms of glycosyl hydrolases.Structure. 1995; 3 (8535779): 853-85910.1016/S0969-2126(01)00220-9Abstract Full Text Full Text PDF PubMed Scopus (1609) Google Scholar, 26Vocadlo D.J. Davies G.J. Mechanistic insights into glycosidase chemistry.Curr. Opin. Chem. Biol. 2008; 12 (18558099): 539-55510.1016/j.cbpa.2008.05.010Crossref PubMed Scopus (336) Google Scholar) (Fig. 4B). In the αG2 complex with the mutant enzyme, Asn-344, which would be Asp-344 in the WT enzyme, is 3.1 Å from the glycosidic oxygen and appropriately positioned to act as a general acid. A water molecule that sits 3.5 Å beneath C1 of the d-galactose residue in the −1 subsite is suitably positioned to be activated as a nucleophile by Asp-321 and/or Asp-345 (Fig. 4B). The GH110 family was initially identified by examination of members that specifically removed the immunodominant α-1,3–linked galactose residues of blood group B antigen. Subsequent characterization of additional GH110 enzymes re-vealed some to be less stringent by possessing the ability to process the linear α-Gal epitope, as well as the blood group B antigen. 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Crystal structure of Aspergillus niger isopullulanase, a member of glycoside hydrolase family 49.J. Mol. Biol. 2008; 376 (18155243): 210-22010.1016/j.jmb.2007.11.098Crossref PubMed Scopus (17) Google Scholar, 34Itoh T. Intuy R. Suyotha W. Hayashi J. Yano S. Makabe K. Wakayama M. Hibi T. Structural
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