Structural Basis of the Catalytic Reaction Mechanism of Novel 1,2-α-L-Fucosidase from Bifidobacterium bifidum
1,2-α-l-Fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucα1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2′-fucosyllactose (substrate), and l-fucose and lactose (products) at 1.12-2.10Å resolution. The AfcA Fuc domain is composed of four regions, an N-terminal β region, a helical linker, an (α/α)6 helical barrel domain, and a C-terminal β region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of l-fucose moiety in 2′-fucosyllactose, and its location is nearly identical near the O1 atom of β-l-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA. 1,2-α-l-Fucosidase (AfcA), which hydrolyzes the glycosidic linkage of Fucα1-2Gal via an inverting mechanism, was recently isolated from Bifidobacterium bifidum and classified as the first member of the novel glycoside hydrolase family 95. To better understand the molecular mechanism of this enzyme, we determined the x-ray crystal structures of the AfcA catalytic (Fuc) domain in unliganded and complexed forms with deoxyfuconojirimycin (inhibitor), 2′-fucosyllactose (substrate), and l-fucose and lactose (products) at 1.12-2.10Å resolution. The AfcA Fuc domain is composed of four regions, an N-terminal β region, a helical linker, an (α/α)6 helical barrel domain, and a C-terminal β region, and this arrangement is similar to bacterial phosphorylases. In the complex structures, the ligands were buried in the central cavity of the helical barrel domain. Structural analyses in combination with mutational experiments revealed that the highly conserved Glu566 probably acts as a general acid catalyst. However, no carboxylic acid residue is found at the appropriate position for a general base catalyst. Instead, a water molecule stabilized by Asn423 in the substrate-bound complex is suitably located to perform a nucleophilic attack on the C1 atom of l-fucose moiety in 2′-fucosyllactose, and its location is nearly identical near the O1 atom of β-l-fucose in the products-bound complex. Based on these data, we propose and discuss a novel catalytic reaction mechanism of AfcA. Bifidobacteria are obligate anaerobic lactic acid-producing bacteria that constitute a substantial fraction of the mammalian intestinal microflora. Interest in bifidobacteria has grown recently following the identification of many beneficial probiotic homeostatic effects, including reducing the presence of harmful bacteria and toxic compounds, immunomodulation, and anticarcinogenic activity (1Bernet M.F. Brassart D. Neeser J.R. Servin A.L. Appl. Environ. Microbiol. 1993; 59: 4121-4128Crossref PubMed Google Scholar, 2Hooper L.V. Gordon J.I. Science. 2001; 292: 1115-1118Crossref PubMed Scopus (1823) Google Scholar, 3Gibson G.R. Wang X. J. Appl. Bacteriol. 1994; 77: 412-420Crossref PubMed Scopus (566) Google Scholar, 4Ouwehand A. Isolauri E. Salminen S. Eur. J. Nutr. 2002; 41: 132-137Crossref PubMed Scopus (203) Google Scholar, 5Femia A.P. Luceri C. Dolara P. Giannini A. Biggeri A. Salvadori M. Clune Y. Collins K.J. Paglierani M. Caderni G. Carcinogenesis. 2002; 23: 1953-1960Crossref PubMed Scopus (294) Google Scholar). Bifidobacteria naturally colonize the lower gastrointestinal tract, but host and microbial factors present in the upper gastrointestinal tract render this environment poor in mono- and disaccharides. Therefore, to survive here, bifidobacteria have evolved the ability to produce a variety of different surface-bound or secreted glycosidases facilitating the metabolism of the diverse sugars found in the lower gastrointestinal tract (6Hoskins L.C. Agustines M. Mcree W.B. Boulding E.T. Kriaris M. Niedermeyer G. J. Clin. Investig. 1985; 75: 944-953Crossref PubMed Scopus (247) Google Scholar, 7Larson G. Falk P. Hoskins L.C. J. Biol. Chem. 1988; 263: 10790-10798Abstract Full Text PDF PubMed Google Scholar, 8Katayama T. Fujita K. Yamamoto K. J. Biosci. Bioeng. 2005; 99: 457-465Crossref PubMed Scopus (61) Google Scholar). There are two broad classes of glycosidases, exo- and endoenzymes, that differ based on the site and mechanism of polysaccharide degradation. Endoglycosidases cleave specific internal glycosidic bonds, but exoglycosidases remove oligosaccharide units at the reducing or nonreducing ends of the polysaccharide chain. Glycosidases can be further divided into two broad families, retaining and inverting glycosidases, according to the stereochemical outcome of their action (9Sincott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1497) Google Scholar, 10McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (819) Google Scholar). Most retaining glycosidases have two catalytic carboxylic acids separated by 5.5 Å in their active site and function through a double displacement mechanism (9Sincott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1497) Google Scholar, 10McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (819) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar). In contrast, inverting glycosidases act through a single displacement mechanism in which the two carboxyl groups, acting as general acid and base catalysts, are ∼10.5 Å apart, allowing simultaneous interactions with a water molecule and substrate (9Sincott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1497) Google Scholar, 10McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (819) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar). Glycosidic cleavage involves the protonation of the glycosidic oxygen by the general acid catalyst in concert with general base-catalyzed nucleophilic attack of a water molecule at the anomeric center. The result is a hemiacetal product with an anomeric configuration that is inverted relative to that of the substrate. 1,2-α-l-Fucosidase (AfcA) 2The abbreviations used are: AfcA, 1,2-α-l-fucosidase; GH, glycoside hydrolase; 2′FL, 2′-fucosyllactose; DFJ, deoxyfuconojirimycin; MES, 2-N-morpholinoethanesulfonic acid; PEG, polyethylene glycol; r.m.s., root mean square. is an exoglycosidase recently identified from Bifidobacterium bifidum (12Katayama T. Sakuma A. Kimura T. Makimura Y. Hiratake J. Sakata K. Yamanoi T. Kumagai H. Yamamoto K. J. Bacteriol. 2004; PubMed Scopus Google Scholar). was classified as a member of the glycoside hydrolase family the on the at B. A. J. PubMed Scopus Google Scholar). AfcA has acids divided an N-terminal domain with a catalytic domain and a C-terminal bacterial domain. The Fuc domain activity to the of from the nonreducing ends of as 2′-fucosyllactose and (12Katayama T. Sakuma A. Kimura T. Makimura Y. Hiratake J. Sakata K. Yamanoi T. Kumagai H. Yamamoto K. J. Bacteriol. 2004; PubMed Scopus Google Scholar). The stereochemical outcome of the l-fucose was determined to be by (12Katayama T. Sakuma A. Kimura T. Makimura Y. Hiratake J. Sakata K. Yamanoi T. Kumagai H. Yamamoto K. J. Bacteriol. 2004; PubMed Scopus Google Scholar). is a present at the ends of of many including and of on the and in J. Biol. Chem. 1985; Full Text PDF PubMed Google Scholar, 2002; PubMed Scopus Google Scholar). of different from bacteria to and the of these the and of l-fucose in in diverse and including 1990; PubMed Scopus Google Scholar, J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Chem. 1995; 3: PubMed Scopus Google and T. Full Text PDF PubMed Scopus Google Scholar). of and in are in many J. T. Kimura M. T. Y. T. A. H. J. PubMed Scopus Google Scholar, E. PubMed Scopus Google Scholar). the presence of in the can be used as an for J. D. A. A. G. Google Scholar, H. A. M. M. H. J. PubMed Scopus Google Scholar, H. T. S. M. K. K. H. Y. T. PubMed Scopus Google Scholar). have used to the function of l-fucose To identified to family based on acid and the from this family is the G. C. T. Withers S.G. Henrissat B. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). However, this is a retaining and the of inverting To better understand the and mechanism of inverting we and of the B. bifidum catalytic domain. of the and the analyses into a novel catalytic mechanism for this and were from and were from and was from and 2′FL, and lactose were from were from and and to the catalytic domain acid of AfcA was by as a (12Katayama T. Sakuma A. Kimura T. Makimura Y. Hiratake J. Sakata K. Yamanoi T. Kumagai H. Yamamoto K. J. Bacteriol. 2004; PubMed Scopus Google and a of with and the was into the site of the were grown in and by the of at for were by at K. The was by the of and was and and the was a and with a of were with and and The was a the with a of active were and a in and The Fuc domain was on a and to in was E. in 1985; PubMed Scopus Google Scholar, J.R. J. 1990; PubMed Scopus Google Scholar). of the was as for the Fuc domain the that in the acid Fuc domain were with The and were as a single on with acid of the AfcA Fuc domain were by for were and as for analyses were as C-terminal and were by the and a Fuc domain was to The and of in the were determined by was used as a and of the activity of the and was at in as a substrate. the reaction were for The of l-fucose was determined by a from a A. J. PubMed Scopus Google Scholar). of was determined in the the of reaction was were determined by of in which substrate was in the of their experiments were by in to a reaction of of and and at K. The reaction was a The was with and l-fucose was by of and Fuc of Fuc domain was grown the from an of in and composed of and polyethylene at The were identical of the complex of the Fuc domain with the were by the in for at K. were a and The complex were by the of with for at K. The of in with l-fucose and lactose were by a and and were at and at at and at the were and the PubMed Scopus Google Scholar). of the was by the the J. D. PubMed Scopus Google at Å resolution. The was by the which acid in the was used for molecular of the the from the D. 1994; PubMed Scopus Google and the were to Å resolution. The of the complex was determined by the molecular a of the as a The structures of the and were determined by the molecular the complex as a The for was the A. Struct. Biol. PubMed Scopus Google Scholar). were from J. Struct. Biol. PubMed Scopus Google Scholar). was from the for the molecule was by the of and the was from to into for 2′FL, and and lactose were from and The of the were with the J. Appl. 1993; Google Scholar). have and no were in the of the and are in were with the J. Appl. Google PubMed Scopus Google on the at and A. B. J. Chem. Scopus Google and of and in are for the in are for the in are for the in are for the of of of and in are for the in are for the in are for the of and of in are for the in a Structural in was used to Fuc domain Structural of the Fuc domain were from the C. PubMed Google and the was used for the these E. K. D. 2004; PubMed Scopus Google Scholar). the crystal structures of the AfcA Fuc domain in the of and in the presence of an and reaction The of the unliganded was determined at Å resolution. The Fuc domain of four an N-terminal β and a helical a helical barrel domain and and a C-terminal β used the to similar to the AfcA Fuc domain, and from family J. H. Structure. 2001; Full Text Full Text PDF PubMed Scopus Google and from family M. Y. M. S. K. T. H. S. Structure. 2004; Full Text Full Text PDF PubMed Scopus Google were highly with root mean of and Å for the and The of was present The catalytic active of of these the helical barrel The N-terminal β of the Fuc domain is composed of in a and is to the four of the N-terminal β has a of to S. S. K. T. Y. T. Y. J. Biol. PubMed Scopus Google T. A. C. J. J. Biol. 2001; PubMed Scopus Google and P. S. G. G. S. E. J. Biol. PubMed Scopus Google but the function of the N-terminal The central helical barrel domain is composed of an (α/α)6 barrel domain, and a identified with the catalytic of inverting of family as from J. Biol. PubMed Scopus Google from M. T. S. S. A. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google and from M. T. S. S. A. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). The for the and of these are and but acid with the helical barrel domain of the AfcA Fuc domain. The (α/α)6 helical is present in and family The molecular of the central helical barrel domain is by a and this probably the is further by the structures of the AfcA Fuc domain with an and The C-terminal β forms a with a of to the C-terminal of and but the function of this The from the at a of Å in revealed buried the a the N-terminal β region, helical linker, and helical barrel and and the oxygen atom of from and water molecule are the ligands for this on the AfcA Fuc domain in and was The of to molecule was as that AfcA Fuc a single a l-fucose is a of mammalian J. Chem. Chem. 1985; Google and is an for the AfcA Fuc domain, with a with the for M. S. K. and K. in To the we the crystal of the AfcA Fuc domain in complex with at Å resolution. The of the the and the complex was but was a these structures in the central helical barrel domain in the helical barrel domain and are in the and the of the central cavity is The molecule is buried in the cavity and a in the crystal The of and in the are by and and the of is by with the The atom of and of the in the molecule through The atom of the molecule forms a with the of the atom of and of with in via a water The and in the molecule are a by the of and carboxyl and are on the of the central In the the position of Glu566 is by Å with the the of the two and are in The atom of Glu566 is separated from the C1 atom of the molecule by Å with DFJ, its is a with The of and Asn423 and are separated by The the C1 atom of the molecule and the two of and Asn423 are and these that a but in the helical barrel domain that the catalytic with crystal of the AfcA Fuc domain in complex with into the acid for its Based on this we acid the and we determined the of the and in the conserved in and to and in the relative to AfcA Fuc domain, but the of these were with the In contrast, of a residue a in but an in with the these that the conserved acids that with are a acid in the conserved in the were and the of the with enzyme, but the for these were with of and Asn423 the that these are for of were identical with that of AfcA, that the of the for the of 2′-fucosyllactose by and of AfcA Fuc in a experiments to further the general in the the residue that acts a general base in a is the of an nucleophilic can the active site and activity Withers S.G. Rev. PubMed Scopus Google Scholar). used as the nucleophilic and the ability of and to l-fucose from the activity of the by at that this residue acts as the general base catalyst for this The product in the reaction was l-fucose but as in analyses that the conserved Glu566 and in the mechanism of AfcA Fuc domain, and we the crystal of the in complex with the substrate at Å to better the molecular of Glu566 in the The crystal two in the and a for the the active site The molecule an at the active were these two with an of The of of the molecule and the was the for molecule was In contrast, molecule was highly similar to the with an of In the crystal the of molecule with the molecule through the for substrate in molecule A. these we that molecule the substrate-bound and we the of molecule The l-fucose moiety of a of by the AfcA Fuc domain is by interactions with the l-fucose and The mechanism of AfcA Fuc domain l-fucose is identical to that of and The atom of forms with the atom of and the of of the in the the of the of the be by at the the the forms a with the of at a of The for is that for which the moiety at its reducing The of the in the G. PubMed Scopus Google for the two was to be which with a the atom of and the atom of the water molecule in forms a with the atom of and is located near the anomeric atom of the glycosidic the structures of the Fuc domain and the complex were the position of the l-fucose moiety at the nonreducing of is identical to that of the molecule the of the of of the acid located in the central In the the atom of forms a with the atom of However, in the substrate-bound this to the and the of is from the anomeric C1 we the crystal of the in complex with reaction at Å resolution. was by the in the presence of and we that the substrate be the of crystal In the molecule is in the and are to l-fucose and lactose in the substrate cavity The the l-fucose moiety a of and β-l-fucose and the of the two were as and by result is with the of the of l-fucose in the environment (12Katayama T. Sakuma A. Kimura T. Makimura Y. Hiratake J. Sakata K. Yamanoi T. Kumagai H. Yamamoto K. J. Bacteriol. 2004; PubMed Scopus Google and that the l-fucose molecule with to crystal l-fucose are present in the The of the and β-l-fucose are identical for the of the of the anomeric The O1 atom of the β-l-fucose is with the atom of at a of Å In contrast, the the O1 atom of and the atom of is the AfcA Fuc domain is an inverting the product of its activity be Therefore, we on the of The mechanism of AfcA Fuc domain reaction product is identical to that of substrate However, two that be by the mutational are in these two The atom of the β-l-fucose moiety forms with the atom of and the atom of Glu566 with the and in the moiety through the and In the substrate-bound the moiety is through a the and the atom of the these two in reaction complex is to for The of the products-bound complex is nearly identical to of the and The of for these structures are and The of Asn423 in the complex a different with its in the complex but this is to the and of the and is an water molecule in and located the atom of and the atom of Asn423 that is probably a mutational The of these different into the catalytic the first crystal of a from family 95. The domain arrangement of the AfcA catalytic domain from that of T. of a composed of a catalytic domain and a C-terminal domain G. C. T. Withers S.G. Henrissat B. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In contrast, the AfcA Fuc domain is similar to bacterial from and which are composed of catalytic an (α/α)6 barrel of helical The (α/α)6 barrel of the Fuc domain is inverting of family in the structures of AfcA Fuc and that the active site of the AfcA Fuc domain is located the in the helical barrel domain, and this was and is the or of the catalytic in inverting but the of the central (α/α)6 domain to into the mechanism of AfcA. In inverting glycosidases, two carboxyl as general acid and base In structures, these are located Å on to the substrate and the nucleophilic water molecule to the catalytic (9Sincott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1497) Google Scholar, 10McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (819) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar). The reaction mechanism of inverting is to be similar to that of the inverting M. Y. M. S. K. T. H. S. Structure. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). In these the nucleophilic is to attack the substrate by the general base the AfcA Fuc domain has a of with the identified which is highly conserved in the is on the catalytic of and inverting to the and The in of these are located in identical and are to act as general acid activity J. H. Structure. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, M. Y. M. S. K. T. H. S. Structure. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google Scholar, M. T. S. S. A. Y. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. E. J. D. PubMed Scopus Google Scholar). In contrast, which we first to be a base catalyst based on the of mutational and is located to the general base in the of the the complex structures and are highly The for of these are Therefore, these structures are identical for the by the mutational effects, and probably the of the the domain of bacterial and J. H. Structure. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. PubMed Scopus Google but we in of the structures with the of the AfcA Fuc domain. that the (α/α)6 domain is for the catalytic the AfcA Fuc domain a this probably the an The single displacement mechanism used by inverting glycosidases on two located carboxyl from acid or acid (9Sincott M.L. Chem. Rev. 1990; 90: 1171-1202Crossref Scopus (1497) Google Scholar, 10McCarter J.D. Withers S.G. Curr. Opin. Struct. Biol. 1994; 4: 885-892Crossref PubMed Scopus (819) Google Scholar, 11Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1628) Google Scholar). However, and mutational analyses revealed a reaction mechanism for AfcA The position of the in the complex is to the a for the atom to the of The activity of this is that Glu566 acts a to the oxygen atom of the glycosidic catalytic base is to the water molecule for nucleophilic attack at the anomeric for inverting However, no carboxylic acid of the catalytic water molecule is found in the of the of the l-fucose residue in the AfcA Fuc domain. acid at position is highly and is located at the of the catalytic to its the water molecule by the substrate in the crystal that to be an for the catalytic However, of with AfcA water molecule forms a with Asn423 and is located at a position for nucleophilic attack of the C1 atom of the l-fucose moiety and The the anomeric C1 atom and the water molecule is and the of this arrangement is with an of as for the complex in inverting the position of this water molecule is nearly identical to that of the O1 atom of the β-l-fucose in the products-bound complex. the of in the complex from its position in the is that the the the atom of with the atom of Therefore, is that this water molecule forms with and Asn423 in for and in the of and the position and of the water allowing the of of the water molecule to the C1 atom of l-fucose moiety of the substrate. In the the atom of forms a with the O1 atom of the β-l-fucose moiety at a of that two of and Asn423 in a from the water molecule through to produce a which in acts as a to attack the C1 atom of the l-fucose the two and are to with the and at of and Å the of water by a of be by these as from the of mutational and experiments with the for the of experiments is that the molecule in the position as in and the of the but this render residue as a catalytic base is that the to acid to and an be Asn423 and a is to is that no reaction was in the or these acids are for the The that involves of the the in S. A. Henrissat B. B. 1995; PubMed Scopus Google Scholar, S. D. B. J. Withers S.G. J. Chem. Scopus Google A. J. Chem. Scopus Google Scholar, J. Chem. Scopus Google Scholar, D. B. S. 2001; PubMed Scopus Google and D. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). In the catalytic of these the of oxygen of of a substrate is and stabilized by the which a with the of the substrate and the oxygen the anomeric to an Withers S.G. Chem. PubMed Google Scholar, Withers S.G. Curr. Opin. Chem. Biol. 4: PubMed Scopus Google Scholar). is that an residue acts as a base a can act in in inverting glycosidases have in a similar reaction mechanism, an acid residue function as a catalytic acid A. G. T. K. M. M. A. D. M.L. M. J. J. Chem. 2001; Scopus Google Scholar, A. J. G. Chem. PubMed Scopus Google Scholar). The catalytic base of of family is near the anomeric center. In this the reaction mechanism is to the of the water via a to the catalytic base inverting of family are to an base catalyst arrangement G. M. C. C. H. J. PubMed Scopus Google Scholar, G. C. C. H. PubMed Scopus Google Scholar, H. J. Biol. 2002; PubMed Scopus Google Scholar). that can as an in a to the of a water and a for and with of their catalytic acid E. Henrissat B. 2005; PubMed Scopus Google Scholar). acids in the and C-terminal of the (α/α)6 domain were to be the catalytic acids of and the C-terminal residue to AfcA residue However, no with the catalytic residue is found in the crystal and and identified an as the general acid catalyst for AfcA. of structures and of the general acid but of AfcA and the general base are that the reaction of family differ from of the and this is the of the cavity family is that these the novel reaction mechanism G. Withers for of the the of the at and at and at for the and for with