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Unique active-site and subsite features in the arabinogalactan-degrading GH43 exo-β-1,3-galactanase from Phanerochaete chrysosporium

2020; Elsevier BV; Volume: 295; Issue: 52 Linguagem: Inglês

10.1074/jbc.ra120.016149

ISSN

1083-351X

Autores

Kaori Matsuyama, N. Kishine, Zui Fujimoto, Naoki Sunagawa, Toshihisa Kotake, Yoichi Tsumuraya, Masahiro Samejima, Kiyohiko Igarashi, Satoshi Kaneko,

Tópico(s)

Polysaccharides and Plant Cell Walls

Resumo

Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization. Arabinogalactan proteins (AGPs) are plant proteoglycans with functions in growth and development. However, these functions are largely unexplored, mainly because of the complexity of the sugar moieties. These carbohydrate sequences are generally analyzed with the aid of glycoside hydrolases. The exo-β-1,3-galactanase is a glycoside hydrolase from the basidiomycete Phanerochaete chrysosporium (Pc1,3Gal43A), which specifically cleaves AGPs. However, its structure is not known in relation to its mechanism bypassing side chains. In this study, we solved the apo and liganded structures of Pc1,3Gal43A, which reveal a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family 35 (CBM35) binding domain. GH43_sub24 is known to lack the catalytic base Asp conserved among other GH43 subfamilies. Our structure in combination with kinetic analyses reveals that the tautomerized imidic acid group of Gln263 serves as the catalytic base residue instead. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Subsite −1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6–linked galactan side chains of AGPs. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s, including those that bind galactomannan. Specifically, we noted a Gly → Trp substitution, which affects pyranose stacking, and an Asp → Asn substitution in the binding pocket, which recognizes β-linked rather than α-linked Gal residues. These findings should facilitate further structural analysis of AGPs and may also be helpful in engineering designer enzymes for efficient biomass utilization. Arabinogalactan proteins (AGPs) are proteoglycans characteristically localized in the plasma membrane, cell wall, and intercellular layer of higher land plants (1Tsumuraya Y. Hashimoto Y. Yamamoto S. Shibuya N. Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves.Carbohydr. Res. 1984; 134: 215-22810.1016/0008-6215(84)85039-9Crossref Scopus (61) Google Scholar), in which they play functional roles in growth and development (2Majewska-Sawka A. Nothnagel E.A. The multiple roles of arabinogalactan proteins in plant development.Plant Physiol. 2000; 122 (10631243): 3-910.1104/pp.122.1.3Crossref PubMed Scopus (273) Google Scholar). The carbohydrate moiety of AGPs is composed of a β-1,3-d-galactan main chain and β-1,6-d-galactan side chain, decorated with arabinose, fucose, and glucuronic acid residues (1Tsumuraya Y. Hashimoto Y. Yamamoto S. Shibuya N. Structure of l-arabino-d-galactan-containing glycoproteins from radish leaves.Carbohydr. Res. 1984; 134: 215-22810.1016/0008-6215(84)85039-9Crossref Scopus (61) Google Scholar, 2Majewska-Sawka A. Nothnagel E.A. The multiple roles of arabinogalactan proteins in plant development.Plant Physiol. 2000; 122 (10631243): 3-910.1104/pp.122.1.3Crossref PubMed Scopus (273) Google Scholar). The chain lengths and frequencies of side chains are different among plant species, organs, and stages of development (3Ellis M. Egelund J. Schultz C.J. Bacic A. Arabinogalactan-proteins: key regulators at the cell surface?.Plant Physiol. 2010; 153 (20388666): 403-41910.1104/pp.110.156000Crossref PubMed Scopus (318) Google Scholar), and the overall structures of the carbohydrate moieties of AGPs are not yet fully understood. Degradation of polysaccharides using specific enzymes is one approach to investigate their structures and roles. In this context, exo-β-1,3-galactanase (EC 3.2.1.145) specifically cleaves the nonreducing end β-1,3–linked galactosyl linkage of β-1,3-galactans to release d-galactose (Gal). In particular, it releases β-1,6-galactooligosaccharides together with Gal from AGPs (4Tsumuraya Y. Mochizuki N. Hashimoto Y. Kovác P. Purification of an Exo-β-(1-3)-d-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins.J. Biol. Chem. 1990; 265 (2158993): 7207-7215Abstract Full Text PDF PubMed Google Scholar, 5Pellerin P. Brillouet J.M. Purification and properties of an exo-(1→3)-β-d-galactanase from Aspergillus niger.Carbohydr. Res. 1994; 264 (7805066): 281-29110.1016/S0008-6215(05)80012-6Crossref PubMed Scopus (30) Google Scholar) and is therefore useful for structural analysis of AGPs. The basidiomycete Phanerochaete chrysosporium produces an exo-β-1,3-galactanase (Pc1,3Gal43A; GenBankTM accession no. BAD98241) that degrades the carbohydrates of AGPs when grown with β-1,3-galactan as a carbon source (6Ichinose H. Yoshida M. Kotake T. Kuno A. Igarashi K. Tsumuraya Y. Samejima M. Hirabayashi J. Kobayashi H. Kaneko S. An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.J. Biol. Chem. 2005; 280 (15866877): 25820-2582910.1074/jbc.M501024200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Pc1,3Gal43A consists of a glycoside hydrolase (GH) family 43 subfamily 24 (GH43_sub24) catalytic domain and a carbohydrate-binding module (CBM) belonging to family 35 (designated as PcCBM6 in (6Ichinose H. Yoshida M. Kotake T. Kuno A. Igarashi K. Tsumuraya Y. Samejima M. Hirabayashi J. Kobayashi H. Kaneko S. An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.J. Biol. Chem. 2005; 280 (15866877): 25820-2582910.1074/jbc.M501024200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar)) based on the amino acid sequences in the Carbohydrate-Active enZymes (CAZy) database (RRID:SCR012935) (6Ichinose H. Yoshida M. Kotake T. Kuno A. Igarashi K. Tsumuraya Y. Samejima M. Hirabayashi J. Kobayashi H. Kaneko S. An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.J. Biol. Chem. 2005; 280 (15866877): 25820-2582910.1074/jbc.M501024200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 7Ishida T. Fujimoto Z. Ichinose H. Igarashi K. Kaneko S. Samejima M. Crystallization of selenomethionyl exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium.Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 2009; 65 (20054127): 1274-127610.1107/S1744309109043395Crossref PubMed Scopus (5) Google Scholar, 8Mewis K. Lenfant N. Lombard V. Henrissat B. Dividing the large glycoside hydrolase family 43 into subfamilies: a motivation for detailed enzyme characterization.Appl. Environ. Microbiol. 2016; 82 (26729713): 1686-169210.1128/AEM.03453-15Crossref PubMed Scopus (98) Google Scholar). The properties of the enzyme have been analyzed using recombinant Pc1,3Gal43A expressed in the methylotrophic yeast Pichia pastoris (6Ichinose H. Yoshida M. Kotake T. Kuno A. Igarashi K. Tsumuraya Y. Samejima M. Hirabayashi J. Kobayashi H. Kaneko S. An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.J. Biol. Chem. 2005; 280 (15866877): 25820-2582910.1074/jbc.M501024200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The CBM35 of Pc1,3Gal43A was characterized as the first β-1,3-galactan–binding module, and Pc1,3Gal43A showed typical GH43_sub24 activity. The enzyme cleaves only β-1,3 linkages of oligosaccharides and polysaccharides but produces β-1,6-galactooligosaccharides together with Gal. Thus, Pc1,3Gal43A specifically recognizes β-1,3–linked Gal but can accommodate β-1,6–bound side chains (6Ichinose H. Yoshida M. Kotake T. Kuno A. Igarashi K. Tsumuraya Y. Samejima M. Hirabayashi J. Kobayashi H. Kaneko S. An exo-β-1,3-galactanase having a novel β-1,3-galactan-binding module from Phanerochaete chrysosporium.J. Biol. Chem. 2005; 280 (15866877): 25820-2582910.1074/jbc.M501024200Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). Glycoside hydrolases are classified into families based on sequence similarity, whereas they are also divided into two major groups according to their catalytic mechanisms (i.e. inverting enzymes and retaining enzymes) (9Davies 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 (1505) Google Scholar, 10Rye C.S. Withers S.G. Glycosidase mechanisms.Curr. Opin. Chem. Biol. 2000; 4 (11006547): 573-58010.1016/S1367-5931(00)00135-6Crossref PubMed Scopus (402) Google Scholar). Inverting enzymes typically utilize two acidic residues that act as an acid and a base, respectively, and a hydroxyl group connected to anomeric carbon inverts from the glycosidic linkage after the reaction. GH43 enzymes are members of the inverting group and share conserved Glu and Asp as the catalytic acid and base, respectively (8Mewis K. Lenfant N. Lombard V. Henrissat B. Dividing the large glycoside hydrolase family 43 into subfamilies: a motivation for detailed enzyme characterization.Appl. Environ. Microbiol. 2016; 82 (26729713): 1686-169210.1128/AEM.03453-15Crossref PubMed Scopus (98) Google Scholar), but GH43_sub24 enzymes lack the catalytic base Asp (8Mewis K. Lenfant N. Lombard V. Henrissat B. Dividing the large glycoside hydrolase family 43 into subfamilies: a motivation for detailed enzyme characterization.Appl. Environ. Microbiol. 2016; 82 (26729713): 1686-169210.1128/AEM.03453-15Crossref PubMed Scopus (98) Google Scholar, 11Cartmell A. McKee L.S. Peña M.J. Larsbrink J. Brumer H. Kaneko S. Ichinose H. Lewis R.J. Viksø-Nielsen A. Gilbert H.J. Marles-Wright J. The structure and function of an arabinan-specific α-1,2-arabinofuranosidase identified from screening the activities of bacterial GH43 glycoside hydrolases.J. Biol. Chem. 2011; 286 (21339299): 15483-1549510.1074/jbc.M110.215962Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Cartmell A. Muñoz-Muñoz J. Briggs J.A. Ndeh D.A. Lowe E.C. Baslé A. Terrapon N. Stott K. Heunis T. Gray J. Yu L. Dupree P. Fernandes P.Z. Shah S. Williams S.J. et al.A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation.Nat. Microbiol. 2018; 3 (30349080): 1314-132610.1038/s41564-018-0258-8Crossref PubMed Scopus (39) Google Scholar). In Ct1,3Gal43A (from Clostridium thermocellum), Glu112 was thought to be the catalytic base (13Jiang D. Fan J. Wang X. Zhao Y. Huang B. Liu J. Zhang X.C. Crystal structure of 1,3Gal43A, an exo-β-1,3-galactanase from Clostridium thermocellum.J. Struct. Biol. 2012; 180 (22960181): 447-45710.1016/j.jsb.2012.08.005Crossref PubMed Scopus (22) Google Scholar), but in BT3683 (from Bacteroides thetaiotamicron), Glu367 (corresponding to Glu112 of Ct1,3Gal43A) was found not to act as a base but to be involved in recognition of the C-4 hydroxyl group of the nonreducing terminal Gal, and instead, Gln577 is predicted to be the catalytic base in the form of an unusual tautomerized imidic acid (12Cartmell A. Muñoz-Muñoz J. Briggs J.A. Ndeh D.A. Lowe E.C. Baslé A. Terrapon N. Stott K. Heunis T. Gray J. Yu L. Dupree P. Fernandes P.Z. Shah S. Williams S.J. et al.A surface endogalactanase in Bacteroides thetaiotaomicron confers keystone status for arabinogalactan degradation.Nat. Microbiol. 2018; 3 (30349080): 1314-132610.1038/s41564-018-0258-8Crossref PubMed Scopus (39) Google Scholar). An example of GH lacking a catalytic base, endoglucanase V from P. chrysosporium (PcCel45A), is already known, and based on the mechanism proposed for this enzyme, it is possible that tautomerized Gln functions as a base in GH43_sub24 or that this Gln stabilizes nucleophilic water. PcCel45A lacks the catalytic base Asp that is conserved in other GH45 subfamilies (14Igarashi K. Ishida T. Hori C. Samejima M. Characterization of an endoglucanase belonging to a new subfamily of glycoside hydrolase family 45 of the basidiomycete Phanerochaete chrysosporium.Appl. Environ. Microbiol. 2008; 74 (18676702): 5628-563410.1128/AEM.00812-08Crossref PubMed Scopus (58) Google Scholar), but it uses the tautomerized imidic acid of Asn as the base, as indicated by neutron crystallography (15Nakamura A. Ishida T. Kusaka K. Yamada T. Fushinobu S. Tanaka I. Kaneko S. Ohta K. Tanaka H. Inaka K. Higuchi Y. Niimura N. Samejima M. Igarashi K. "Newton's cradle" proton relay with amide–imidic acid tautomerization in inverting cellulase visualized by neutron crystallography.Sci. Adv. 2015; 1 (26601228)e150026310.1126/sciadv.1500263Crossref PubMed Scopus (51) Google Scholar). However, it is difficult to understand the situation in GH43_sub24, because no holo structure with a ligand at the catalytic center has yet been solved in this family. Moreover, no structure of eukaryotic GH43_sub24 has yet been reported. The CBM35 module is composed of ∼140 amino acids. This family includes modules with various binding characteristics and decorated with xylans, mannans, β-1,3-galactans, and glucans (16Montanier C. Van Bueren A.L. Dumon C. Flint J.E. Correia M.A. Prates J.A. Firbank S.J. Lewis R.J. Grondin G.G. Ghinet M.G. Gloster T.M. Herve C. Knox J.P. Talbot B.G. Turkenburg J.P. et al.Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.Proc. Natl. Acad. Sci. U. S. A. 2009; 106 (19218457): 3065-307010.1073/pnas.0808972106Crossref PubMed Scopus (87) Google Scholar, 17Correia M.A.S. Abbott D.W. Gloster T.M. Fernandes V.O. Prates J.A.M. Montanier C. Dumon C. Williamson M.P. Tunnicliffe R.B. Liu Z. Flint J.E. Davies G.J. Henrissat B. Coutinho P.M. Fontes C.M.G.A. et al.Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.Biochemistry. 2010; 49 (20496884): 6193-620510.1021/bi1006139Crossref PubMed Scopus (31) Google Scholar, 18Couturier M. Roussel A. Rosengren A. Leone P. Stålbrand H. Berrin J.G. Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis.J. Biol. Chem. 2013; 288 (23558681): 14624-1463510.1074/jbc.M113.459438Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Okazawa Y. Miyazaki T. Yokoi G. Ishizaki Y. Nishikawa A. Tonozuka T. Crystal structure and mutational analysis of isomalto-dextranase, a member of glycoside hydrolase family 27.J. Biol. Chem. 2015; 290 (26330557): 26339-2634910.1074/jbc.M115.680942Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 20Fujimoto Z. Kishine N. Suzuki N. Suzuki R. Mizushima D. Momma M. Kimura K. Funane K. Isomaltooligosaccharide-binding structure of Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase.Biosci. Rep. 2017; 37 (28385816)BSR2017025310.1042/bsr20170253Crossref PubMed Scopus (7) Google Scholar, 21Suzuki N. Fujimoto Z. Kim Y.M. Momma M. Kishine N. Suzuki R. Suzuki S. Kitamura S. Kobayashi M. Kimura A. Funane K. Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.J. Biol. Chem. 2014; 289 (24616103): 12040-1205110.1074/jbc.M114.547992Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The family members are divided into four clusters based on their sequences and binding specificities (17Correia M.A.S. Abbott D.W. Gloster T.M. Fernandes V.O. Prates J.A.M. Montanier C. Dumon C. Williamson M.P. Tunnicliffe R.B. Liu Z. Flint J.E. Davies G.J. Henrissat B. Coutinho P.M. Fontes C.M.G.A. et al.Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.Biochemistry. 2010; 49 (20496884): 6193-620510.1021/bi1006139Crossref PubMed Scopus (31) Google Scholar). The structures of CBM35s binding with xylan, mannan, and glucan have already been solved (16Montanier C. Van Bueren A.L. Dumon C. Flint J.E. Correia M.A. Prates J.A. Firbank S.J. Lewis R.J. Grondin G.G. Ghinet M.G. Gloster T.M. Herve C. Knox J.P. Talbot B.G. Turkenburg J.P. et al.Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.Proc. Natl. Acad. Sci. U. S. A. 2009; 106 (19218457): 3065-307010.1073/pnas.0808972106Crossref PubMed Scopus (87) Google Scholar, 17Correia M.A.S. Abbott D.W. Gloster T.M. Fernandes V.O. Prates J.A.M. Montanier C. Dumon C. Williamson M.P. Tunnicliffe R.B. Liu Z. Flint J.E. Davies G.J. Henrissat B. Coutinho P.M. Fontes C.M.G.A. et al.Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.Biochemistry. 2010; 49 (20496884): 6193-620510.1021/bi1006139Crossref PubMed Scopus (31) Google Scholar, 18Couturier M. Roussel A. Rosengren A. Leone P. Stålbrand H. Berrin J.G. Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis.J. Biol. Chem. 2013; 288 (23558681): 14624-1463510.1074/jbc.M113.459438Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Okazawa Y. Miyazaki T. Yokoi G. Ishizaki Y. Nishikawa A. Tonozuka T. Crystal structure and mutational analysis of isomalto-dextranase, a member of glycoside hydrolase family 27.J. Biol. Chem. 2015; 290 (26330557): 26339-2634910.1074/jbc.M115.680942Abstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar, 20Fujimoto Z. Kishine N. Suzuki N. Suzuki R. Mizushima D. Momma M. Kimura K. Funane K. Isomaltooligosaccharide-binding structure of Paenibacillus sp. 598K cycloisomaltooligosaccharide glucanotransferase.Biosci. Rep. 2017; 37 (28385816)BSR2017025310.1042/bsr20170253Crossref PubMed Scopus (7) Google Scholar, 21Suzuki N. Fujimoto Z. Kim Y.M. Momma M. Kishine N. Suzuki R. Suzuki S. Kitamura S. Kobayashi M. Kimura A. Funane K. Structural elucidation of the cyclization mechanism of α-1,6-glucan by Bacillus circulans T-3040 cycloisomaltooligosaccharide glucanotransferase.J. Biol. Chem. 2014; 289 (24616103): 12040-1205110.1074/jbc.M114.547992Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar), but no structure of β-1,3-galactan–binding CBM35 has yet been reported. In the present work, we solved the apo and liganded structures of Pc1,3Gal43A. Based on the results, we discuss the catalytic mechanism and the mode of ligand binding to CBM35 in the two-domain structure. The crystal structure of the SeMet derivative of Pc1,3Gal43A was first determined by means of the multiwavelength anomalous dispersion method, and this was followed by structure determination of the ligand-free WT, the WT bound with Gal (WT_Gal), the E208Q mutant co-crystallized with β-1,3-galactotriose (Gal3; E208Q_Gal3), and the E208A mutant co-crystallized with Gal3 (E208A_Gal3). Data collection statistics and structural refinement statistics are summarized in Table 1, Table 2, respectively.Table 1Data collection statisticsDataWTSeMetWT Gal3 soakingE208Q Gal3 co-crystalE208A Gal3 co-crystalPeakEdgeLow remoteHigh remoteSpace groupP1P21P21P21P21P212121P21P3221Unit-cell parameters a, b, c (Å)40.5, 66.3, 74.066.4, 50.5, 75.850.8, 66.6, 106.466.1, 50.4, 75.7156.7, 156.7, 147.7 α, β, γ (degrees)72.0, 84.7, 82.190.0, 111.9, 90.090.0, 90.0, 90.090.0, 111.3, 90.090.0, 120.0, 90.0Beam linePF BL-5PF BL-6APF BL-6APF BL-6APF BL-6APF-AR NW12PF-AR NE3PF-AR NE3DetectorADSC Q315ADSC Q4RADSC Q210ADSC Q270ADSC Q270Wavelength (Å)0.906460.978820.979500.983000.964001.00001.00001.0000Resolution (Å)50–1.40(1.45–1.40)50.0–1.80 (1.86–1.80)50.0–2.00 (2.07–2.00)50.0–2.00 (2.07–2.00)50.0–2.00 (2.07–2.00)100.0–1.50 (1.55–1.50)50.0–2.50 (2.54–2.50)100.0–2.30 (2.38–2.30)Rsym0.054 (0.370)0.079 (0.672)0.061 (0.307)0.060 (0.303)0.062 (0.307)0.046 (0.109)0.143 (0.399)0.167 (0.627)Completeness (%)95.6 (89.0)100.0 (99.9)100.0 (100.0)100.0 (100.0)100.0 (100.0)97.5 (94.9)96.2 (83.0)99.1 (92.0)Multiplicity3.8 (3.1)14.0 (12.6)7.2 (6.9)7.2 (6.9)7.2 (7.0)9.2 (8.9)4.4 (3.0)9.7 (5.1)Average I/σ(I)24.4 (2.8)36.6 (4.7)30.9 (8.3)30.8 (8.2)31.3 (8.2)48.9 (21.0)13.5 (2.7)17.9 (2.7)Unique reflections136,692 (12,747)43,643 (4,353)31,744 (3,139)31,760 (3,144)31,780 (3,146)57,278 (5,493)16,007 (702)92,497 (8,510)Observed reflections520,085613,162227,158228,381228,595524,95769,939900,469Z21114 Open table in a new tab Table 2Refinement statisticsDataWTWT_GalE208Q_Gal3E208A_Gal3Resolution range7.997–1.398 (1.448–1.398)41.56–1.500 (1.554–1.500)29.79–2.499 (2.588–2.499)30.66–2.300 (2.382–2.300)Completeness (%)95.46 (87.82)97.51 (94.80)96.41 (85.67)98.78 (92.17)Wilson B-factor12.7610.1129.9130.40Reflections used in refinement136,655 (12,497)57,105 (5,474)15,762 (1,381)92,011 (8,507)Reflections used for R-free6,862 (630)2,884 (272)799 (64)4,568 (441)R-work (%)15.47 (22.50)13.43 (12.71)16.62 (25.54)16.10 (22.39)R-free (%)18.56 (26.28)16.00 (17.93)24.39 (42.53)21.43 (28.28)No. of nonhydrogen atoms7,9663,9233,57614,570 Macromolecules6,6153,2903,23512,886 Ligands109121114678 Solvent1,2425122271,006Protein residues2,1064274281,708r.m.s. (bonds)0.0080.0060.0080.011r.m.s. (angles)1.220.870.941.05Ramachandran favored (%)97.2997.4194.1395.76Ramachandran allowed (%)2.712.595.874.24Ramachandran outliers (%)0000Rotamer outliers (%)0.810.550.290.36Clash score2.061.956.943.50Average B-factor (Å2)17.2112.4530.4832.98 Macromolecules14.9710.5729.7731.60 Ligands29.3823.3352.2656.11 Solvent28.0922.0229.7435.03PDB code7BYS7BYT7BYV7BYX Open table in a new tab The recombinant Pc1,3Gal43A molecule is composed of a single polypeptide chain of 428 amino acids (Gln21–Tyr448) with two extra amino acids, Glu19 and Phe20, derived from the restriction enzyme cleavage site, which are disordered and thus were not observed. The protein is decorated with N-glycans because it was expressed in Pichia yeast. Up to three sugar chains are attached at Asn79, Asn194, and Asn389; the attached chains vary in position and structure, and most contain one or two GlcNAc moieties. Pc1,3Gal43A is composed of two domains, and ligands introduced by soaking or co-crystallization are located in a subsite of the catalytic domain or the binding site of CBM35 (Fig. 1). The N-terminal catalytic domain consists of a five-bladed β-propeller (Gln21–Gly325), as in other GH clan-F enzymes, and the C-terminal domain (PcCBM35) takes a β-jellyroll fold (Thr326–Tyr448) structure, as in previously reported CBM35s (16Montanier C. Van Bueren A.L. Dumon C. Flint J.E. Correia M.A. Prates J.A. Firbank S.J. Lewis R.J. Grondin G.G. Ghinet M.G. Gloster T.M. Herve C. Knox J.P. Talbot B.G. Turkenburg J.P. et al.Evidence that family 35 carbohydrate binding modules display conserved specificity but divergent function.Proc. Natl. Acad. Sci. U. S. A. 2009; 106 (19218457): 3065-307010.1073/pnas.0808972106Crossref PubMed Scopus (87) Google Scholar, 17Correia M.A.S. Abbott D.W. Gloster T.M. Fernandes V.O. Prates J.A.M. Montanier C. Dumon C. Williamson M.P. Tunnicliffe R.B. Liu Z. Flint J.E. Davies G.J. Henrissat B. Coutinho P.M. Fontes C.M.G.A. et al.Signature active site architectures illuminate the molecular basis for ligand specificity in family 35 carbohydrate binding module.Biochemistry. 2010; 49 (20496884): 6193-620510.1021/bi1006139Crossref PubMed Scopus (31) Google Scholar, 18Couturier M. Roussel A. Rosengren A. Leone P. Stålbrand H. Berrin J.G. Structural and biochemical analyses of glycoside hydrolase families 5 and 26 β-(1,4)-mannanases from Podospora anserina reveal differences upon manno-oligosaccharide catalysis.J. Biol. Chem. 2013; 288 (23558681): 14624-1463510.1074/jbc.M113.459438Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 19Okazawa Y. Miyazaki T. Yokoi G. Ishizaki Y. Nishikawa A. Tonozuka T. Crystal structure and mutational analysis of isomalto-dextranase, a member of glycoside hydrolase family 27.J. Biol. 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De Morais M.A.B. De Lima E.A. Oliveira L. Persinoti G.F. Murakami M.T. Spatially remote motifs cooperatively affect substrate preference of a ruminal GH26-type endo-β-1,4-mannanase.J. Biol. Chem. 2020; 295 (32139511): 5012-502110.1074/jbc.RA120.012583Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar). PcCBM35 contains one calcium ion near the end of the first β-strand on a different domain surface from the plane to which the ligand binds (Fig. 1). The structure of PcCBM35 is similar to those of other known CBM35s. The interface area is 686 Å2 and includes many water molecules. The PDBePISA server (RRID:SCR015749) indicates that the enzyme forms a complex in the crystal, but this is an effect of crystallization, and the enzyme exists as a monomer in solution (data not shown). The five-bladed β-propeller exhibits an almost spherical structure, and two central cavities are located at the ends of the pseudo-5-fold axis (Fig. 1). One of them contains the catalytic site and it is common in almost all GH43 enzymes. The catalytic site is located in the center of the five-bladed β-propeller, whose blades are formed by Gln21 or Asn22–Leu87 (I in Fig. 1), Ser88–Asp155 (II in Fig. 1), Ser156–Gly204 (III in Fig. 1), Ala205–Ser247 (IV in Fig. 1), and Ala248–Asp297 (V in Fig. 1). As shown in Fig. 2, the Gal3 molecule co-crystallized with the E208Q mutant occupies subsites −1, +1, and +2 of the catalytic site, from the nonreducing end to the reducing end. Gal−1 is located at the bottom of the catalytic cavity, and Gal+1 and Gal+2 extend linearly outwards. Gal+1 is half-buried in the cavity, whereas Gal+2 is exposed at the surface (Fig. 2A). Gal−1 adopts a 1S3 skew boat conformation and interacts with many residues via hydrogen bonds and hydrophobic interactions. As shown in Fig. 2 (B and C), the C-2 hydroxyl group of Gal−1 forms hydrogen bonds with NH2 of Arg103 and with OE1 of Gln263 via water. In addition, this water molecule is bound with O of Gly228. The C-3 hydroxyl group of Gal−1 also forms a hydrogen bond with OE2 of Glu57 via water. Glu102, Tyr126, Asp158, Gln208, Thr226, Trp229, and Gln263 interact with Gal3 through hydrophobic interactions. Notably, Trp229 supports the flat C3-C4-C5-C6 structure of Gal−1, and Tyr126 recognizes the C-6 methylol and C-4 hydroxyl groups, whereas Glu102 recognizes the C-3 hydroxyl and C-4 hydroxyl g

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