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• W2076261290 abstract "Fucosylated glycoconjugates are involved in numerous biological events, and α-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in α-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into α-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima α-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)8-like domain and a C-terminal β-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp244, and the Brønsted acid/base, Glu266. Because T. maritima α-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis. Fucosylated glycoconjugates are involved in numerous biological events, and α-l-fucosidases, the enzymes responsible for their processing, are therefore of crucial importance. Deficiency in α-l-fucosidase activity is associated with fucosidosis, a lysosomal storage disorder characterized by rapid neurodegeneration, resulting in severe mental and motor deterioration. To gain insight into α-l-fucosidase function at the molecular level, we have determined the crystal structure of Thermotoga maritima α-l-fucosidase. This enzyme assembles as a hexamer and displays a two-domain fold, composed of a catalytic (β/α)8-like domain and a C-terminal β-sandwich domain. The structures of an enzyme-product complex and of a covalent glycosyl-enzyme intermediate, coupled with kinetic and mutagenesis studies, allowed us to identify the catalytic nucleophile, Asp244, and the Brønsted acid/base, Glu266. Because T. maritima α-l-fucosidase occupies a unique evolutionary position, being far more closely related to the mammalian enzymes than to any other prokaryotic homolog, a structural model of the human enzyme was built to document the structural consequences of the genetic mutations associated with fucosidosis. α-l-Fucosidases catalyze the removal of nonreducing terminal l-fucose residues linked via α-1,2, α-1,3, α-1,4, or α-1,6 bonds to oligosaccharides and their conjugates. Decreased α-l-fucosidase activity is related to a number of pathological conditions such as inflammation, cancer, and cystic fibrosis (1Johnson S.W. Alhadeff J.A. Comp. Biochem. Physiol. B. 1991; 99: 479-488Crossref PubMed Scopus (77) Google Scholar, 2Staudacher E. Altmann F. Wilson I.B. Marz L. Biochim. Biophys. Acta. 1999; 1473: 216-236Crossref PubMed Scopus (196) Google Scholar, 3Becker D.J. Lowe J.B. Glycobiology. 2003; 13: 41R-53RCrossref PubMed Scopus (654) Google Scholar). Severe deficiency of α-l-fucosidases causes fucosidosis, an autosomal recessive lysosomal storage disease resulting in the lethal accumulation of fucosylated glycoconjugates in the lysosomes of most tissues, including the peripheral and central nervous systems (4Willems P.J. Seo H.C. Coucke P. Tonlorenzi R. O'Brien J.S. Eur. J. Hum. Genet. 1999; 7: 60-67Crossref PubMed Scopus (73) Google Scholar). α-l-Fucosidases are also of considerable interest because fucosidase activity in serum can be used in the diagnosis of patients with early colorectal and hepatocellular cancers (5Hutchinson W.L. Johnson P.J. Du M.Q. Williams R. Clin. Sci. 1991; 81: 177-182Crossref Scopus (29) Google Scholar). α-l-Fucosidases have received much attention due to the central role of fucosylated glycoconjugates in many biological events. The latter are widely distributed in the animal kingdom, where they can be encountered in a variety of tissues, including liver, brain, and spleen, in human amniotic fluid, and as a discriminative feature of ABO and Lewis blood group antigens (6Greenwell P. Glycoconj. J. 1997; 14: 159-173Crossref PubMed Scopus (129) Google Scholar). Accordingly, fucosylated glycans play a crucial role in many physiological and pathological processes, including immune response (7Delves P.J. Autoimmunity. 1998; 27: 239-253Crossref PubMed Scopus (44) Google Scholar), signal transduction (8Moloney D.J. Shair L.H. Lu F.M. Xia J. Locke R. Matta K.L. Haltiwanger R.S. J. Biol. Chem. 2000; 275: 9604-9611Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar), development (9Xiang J. Bernstein I.A. Biochem. Biophys. Res. Commun. 1992; 189: 27-32Crossref PubMed Scopus (7) Google Scholar), and adhesion processes of pathogens (10Hooper L.V. Gordon J.I. Glycobiology. 2001; 11: 1R-10RCrossref PubMed Scopus (204) Google Scholar). Implications of fucose in sperm-egg interaction (11Sinowatz F. Plendl J. Kolle S. Acta Anat. 1998; 161: 196-205Crossref PubMed Scopus (33) Google Scholar), early embryogenesis (12Solter D. Knowles B.B. Proc. Natl. Acad. Sci. U. S. A. 1978; 75: 5565-5569Crossref PubMed Scopus (1123) Google Scholar), and apoptosis (13Hiraishi K. Suzuki K. Hakomori S. Adachi M. Glycobiology. 1993; 3: 381-390Crossref PubMed Scopus (151) Google Scholar) have been reported, and alterations in the fucosylation pattern have been observed in a number of diseases, including cancer and diabetes (14Muramatsu T. Glycobiology. 1993; 3: 291-296Crossref PubMed Scopus (140) Google Scholar, 15Wiese T.J. Dunlap J.A. Yorek M.A. Biochim. Biophys. Acta. 1997; 1335: 61-72Crossref PubMed Scopus (44) Google Scholar). Fucose also appears to be an important immune modulator for the interactions between the selectin family of cell adhesion molecules and the sialyl Lewis X antigen of their glycoconjugate counterreceptors, enabling the rolling of leukocytes on the endothelium and subsequent extravasation (16Lowe J.B. Immunol. Rev. 2002; 186: 19-36Crossref PubMed Scopus (199) Google Scholar). α-l-Fucosidases are found exclusively in family GH29 of the classification of glycosidases based on sequence similarities, which reflect the folds, active site architecture, molecular mechanism, and, to a minor extent, substrate specificity (17Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2574) Google Scholar). 1Available at afmb.cnrs-mrs.fr/CAZY. 1Available at afmb.cnrs-mrs.fr/CAZY. α-l-Fucosidases have been shown to follow a double displacement mechanism with net retention of the anomeric configuration, as initially proposed for human liver α-l-fucosidase in 1987 (18White Jr., W.J. Schray K.J. Legler G. Alhadeff J.A. Biochim. Biophys. Acta. 1987; 912: 132-138Crossref PubMed Scopus (17) Google Scholar) and recently confirmed by biochemical studies on α-l-fucosidases from Thermus sp. Y5 (19Eneyskaya E.V. Kulminskaya A.A. Kalkkinen N. Nifantiev N.E. Arbatskii N.P. Saenko A.I. Chepurnaya O.V. Arutyunyan A.V. Shabalin K.A. Neustroev K.N. Glycoconj. J. 2001; 18: 827-834Crossref PubMed Scopus (27) Google Scholar), the marine mollusc Pecten maximus (20Berteau O. McCort I. Goasdoue N. Tissot B. Daniel R. Glycobiology. 2002; 12: 273-282Crossref PubMed Scopus (67) Google Scholar), Sulfolobus solfataricus (21Cobucci-Ponzano B. Trincone A. Giordano A. Rossi M. Moracci M. Biochemistry. 2003; 42: 9525-9531Crossref PubMed Scopus (43) Google Scholar), and Thermotoga maritima (22Tarling C.A. He S. Sulzenbacher G. Bignon C. Bourne Y. Henrissat B. Withers S.G. J. Biol. Chem. 2003; 278: 47394-47399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). We have determined the crystal structure of the α-l-fucosidase from the marine hyperthermophilic bacterium T. maritima (TM aFuc). 2The abbreviations used are: TM aFuc, T. maritima α-l-fucosidase; pNP-Fuc, p-nitrophenyl fucoside; MAD, multiple anomalous dispersion. 2The abbreviations used are: TM aFuc, T. maritima α-l-fucosidase; pNP-Fuc, p-nitrophenyl fucoside; MAD, multiple anomalous dispersion. Currently, this enzyme is the closest bacterial relative of mammalian α-l-fucosidases and shares 38% identity with its human counterpart. The true biological role of TM aFuc and its natural substrate is not known. However, this bacterium appears to have evolved a remarkably large repertoire of enzymes dedicated to the breakdown of plant pectin and xyloglucan. These hemicelluloses often carry α-l-fucose side chains, and it is thus tempting to speculate that TM aFuc operates synergistically with other glycosidases to degrade these polymers. Alternatively, it is also conceivable that TM aFuc may participate in the degradation of algal fucoidan by removing α-1,2- and α-1,4-linked fucosyl side chains from the polysaccharide. The crystal structures of complexes of TM aFuc with α-l-fucose and with a mechanism-based inhibitor, 2-deoxy-2-fluoro-α-l-fucopyranosyl fluoride (fucosyl fluoride), allowed us to delineate the substrate-binding pocket and the covalent 2-deoxy-2-fluoro-l-fucosyl-enzyme intermediate. Together with kinetic and mutagenesis experiments, these structures reveal for the first time the overall fold of glycoside hydrolase family GH29, allow the identification of the two catalytic residues, and provide a structural template to understand the molecular basis of fucosidosis. Cloning—The coding sequence of the T. maritima gene (TM0306) was amplified by PCR from genomic DNA, and the PCR product was subcloned into the pDEST17 prokaryotic expression vector (Gateway, Invitrogen) following the manufacturer's instructions. This vector encodes an N-terminal His6 tag fused to the entire coding region of α-l-fucosidase. Sequence analysis revealed a single base difference compared with GenBank™/EBI accession number NC_000853, which resulted in a Y271C mutation. Expression and Purification—Expression was carried out using Escherichia coli BL21(DE3) cells grown in LB medium at 37 °C to A600 = 0.6 and induced by the addition of 0.5 mm isopropyl-1-thio-β-d-galactopyranoside. After incubation at 37 °C for 4 h, the cells were sedimented, resuspended in 200 mm NaCl and 20 mm Tris-HCl (pH 8.0), and lysed with a French press. After centrifugation, the supernatant was incubated for 15 min at 60 °C, and precipitated E. coli proteins were eliminated by centrifugation. The enzyme was further purified by Ni2+ affinity chromatography, and a gel filtration step yielded highly purified enzyme as judged by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Selenomethionine-substituted enzyme was produced using the same bacterial strain grown in minimal M9 medium and supplemented, before induction, with selenomethionine and amino acids known to inhibit methionine biosynthesis (23Van Duyne G.D. Standaert R.F. Karplus P.A. Schreiber S.L. Clardy J. J. Mol. Biol. 1993; 229: 105-124Crossref PubMed Scopus (1073) Google Scholar). Site-directed Mutagenesis and Kinetic Assays—The E66A, D224A, and E266A mutants were generated using the QuikChange™ mutagenesis kit (Stratagene) according to the manufacturer's instructions and were verified by DNA sequencing. These mutants, expressed under the conditions described above, were insoluble, and the coding sequences of the mutants, together with that of wild-type TM aFuc as a control, were therefore subcloned from pDEST17 into the expression vector His-pKM596, a derivative of pKM596 (24Fox J.D. Routzahn K.M. Bucher M.H. Waugh D.S. FEBS Lett. 2003; 537: 53-57Crossref PubMed Scopus (93) Google Scholar), resulting in four His-maltose-binding protein-α-l-fucosidase-pKM596 expression constructs. Expression and purification of these uncleaved fusion proteins were carried out as described above for the wild-type TM aFuc construct, and Michaelis-Menten parameters for the hydrolysis of p-nitrophenyl fucoside (pNP-Fuc) and fucosyl fluoride were obtained as described previously (22Tarling C.A. He S. Sulzenbacher G. Bignon C. Bourne Y. Henrissat B. Withers S.G. J. Biol. Chem. 2003; 278: 47394-47399Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). kcat/Km values were determined from initial rates at low substrate concentrations through Lineweaver-Burk analysis. Crystallization and Data Collection—Crystals were grown by the hanging-drop vapor diffusion method at 20 °C from protein solution at 5.0 mg/ml mixed with 18% (w/v) polyethylene glycol 6000, 100 mm Tris-HCl (pH 8.0), and 5% Jeffamine M-600. Crystals belong to space group H32 and contain two molecules/asymmetric unit. Crystals of the fucose complex and the inhibitor complex were obtained by short soaks in the crystallization solution supplemented with 0.5 m fucose and/or a small amount of inhibitor powder, respectively. All data sets were collected at 100 K on flash-frozen crystals. Cryosolutions were of the same composition as the crystallization/harvesting solutions with the addition of increasing amounts of Jeffamine M-600 and supplemented with 5% (v/v) glycerol in the case of native, selenomethionylated, and inhibitor complex crystals and with the addition of 0.5 m fucose in the case of the fucose complex. Data were indexed and integrated with DENZO (25Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar), and all further computing was carried out with the CCP4 program suite (26CCP4 Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar), unless otherwise stated. Data collection statistics are summarized in Table I.Table IData collection and refinement statisticsMAD (Se)NativeFucoseIntermediateEdgePeakRemoteData collectionBeam lineESRF-ID29ESRF-ID29ESRF-ID29ESRF-EH2ESRF-EH2ESRF-EH1Wavelength (Å)0.97940.97920.86700.9330.9330.934a = b, c (Å)176.50, 172.23176.70, 172.70176.50, 172.39172.46, 167.41179.08, 174.69172.16, 167.33Resolution range (Å)aValues in parentheses are for the highest resolution shell.38-2.50 (2.64-2.50)38-2.50 (2.64-2.50)38-2.50 (2.64-2.50)37-2.40 (2.46-2.40)57-2.80 (2.87-2.80)55-2.25 (2.31-2.25)RmergeaValues in parentheses are for the highest resolution shell.bRmerge=∑hkl∑i|Ihkli-〈Ihkli〉|/∑hkl∑i〈Ihkli〉.0.112 (0.434)0.114 (0.444)0.105 (0.285)0.069 (0.416)0.056 (0.442)0.065 (0.397)RanomaValues in parentheses are for the highest resolution shell.cRanom=∑|〈I+〉-〈I-〉|/∑(〈I+〉+〈I-〉).0.055 (0.148)0.057 (0.138)0.052 (0.100)No. of observations288,280389,051269,165212,59899,148156,795No. of unique reflections35,89036,02535,85636,83324,59837,238Completeness (%)aValues in parentheses are for the highest resolution shell.99.9 (99.9)99.9 (99.6)99.9 (99.9)99.3 (99.3)98.1 (98.1)89.4 (89.4)RedundancyaValues in parentheses are for the highest resolution shell.8.0 (8.1)10.7 (10.8)7.4 (7.5)5.7 (5.4)3.9 (3.9)4.2 (4.0)〈I/σI〉aValues in parentheses are for the highest resolution shell.4.1 (1.2)4.1 (1.6)3.1 (0.8)8.9 (1.8)6.8 (1.6)7.1 (1.8)B from Wilson statistics (Å2)54.3755.6652.5447.93100.8140.90RefinementResolution (Å)37-2.420-2.820-2.25No. of protein atomsdPer asymmetric unit, corresponding to two molecules of TM aFuc.703870047030No. of water molecules/ligand atomsdPer asymmetric unit, corresponding to two molecules of TM aFuc.162/--/22209/42Rcryst/Rfree (%)eRcryst=∑||Fo-Fc||/∑|Fo|.18.52/22.8420.11/23.0318.19/22.99r.m.s. 1-2 bond distances (Å)0.008 (0.020)0.007 (0.020)0.011 (0.020)r.m.s. 1-3 bond angles1.10° (1.9°)0.99° (1.9°)1.21° (1.9°)Average main/side chain B (Å2)60.27/60.7495.05/95.9640.12/41.93Average B Solvent/ligand (Å2)51.09/--/90.040.3/37.8Main chain Δ B, bonded atoms (Å2)1.0620.9891.094a Values in parentheses are for the highest resolution shell.b Rmerge=∑hkl∑i|Ihkli-〈Ihkli〉|/∑hkl∑i〈Ihkli〉.c Ranom=∑|〈I+〉-〈I-〉|/∑(〈I+〉+〈I-〉).d Per asymmetric unit, corresponding to two molecules of TM aFuc.e Rcryst=∑||Fo-Fc||/∑|Fo|. Open table in a new tab Structure Solution and Refinement—The structure of α-fucosidase was solved by the selenomethionine three-wavelength multiple anomalous dispersion (MAD) method using the program SOLVE (27Terwilliger T.C. Berendzen J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 849-861Crossref PubMed Scopus (3216) Google Scholar). The initial MAD phases had mean figures of merit of 0.45 for data to 2.8-Å resolution and of 0.62 after 10 cycles of density modification using DM, implementing solvent flattening and histogram matching (28Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar). Despite the fact that only 6 selenium atoms of 12 could be located, with 6 selenomethionine residues being located either at the N terminus or in disordered loop regions, the resulting experimental map was of good quality and could be further improved by NCS averaging and phase combination techniques. Both protein chains were manually traced and fitted with the program TURBO-FRODO (29Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics Corp., 1991: 81Google Scholar). Refinement was carried out against data of the native crystal with CNS (30Bruenger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) and REFMAC (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar) using the maximum likelihood approach and incorporating bulk solvent corrections, anisotropic Fo versus Fc scaling, NCS restraints, and TLS refinement. Weak data in the outmost resolution shell were included in the refinement, as they improved considerably sigmaA weighted electron density maps. A random 7% (2642) of reflections were set aside for cross-validation purposes. BUSTER (32Roversi P. Blanc E. Vonrhein C. Evans G. Bricogne G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2000; 56: 1316-1323Crossref PubMed Scopus (69) Google Scholar) was used to determine ill defined regions of the model. Automated solvent building was performed with ARP/wARP (33Perrakis A. Morris R. Lamzin V.S. Nat. Struct. Biol. 1999; 6: 458-463Crossref PubMed Scopus (2561) Google Scholar). The two complex structures were subjected to rigid body refinement with REFMAC (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar), and further refinement was carried out as described above. Ligands were included only toward the end of refinement, and no torsion angle restraints were applied to avoid imposition of a given pyranose ring conformation. The two molecules present in the asymmetric unit encompass Arg7-Pro46, Asp56-His268, and Asp274-Val447. Six N-terminal residues as well as the N-terminal His6 tag and the linker used for cloning and purification, 2 C-terminal residues, and surface loops Thr47-Met55, Val269-Gly273, and Gly297-His300 in chain B could not be built due to either diffuse or totally absent electron density. These surface regions, devoid of crystal-packing interactions, were also missing in the structures of the l-fucose complex and the glycosyl-enzyme intermediate. Two solvent-exposed Cys residues, Cys364 and Cys365, form a rare disulfide bridge between consecutive residues. The linked l-fucosyl moiety and the product refined to occupancies of 0.4 and 0.6, respectively, in one molecule of the asymmetric unit and 0.6 and 0.4, respectively, in the second molecule. The stereochemistry of the final models was verified with the program PROCHECK (34Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Refinement and structure quality statistics are listed in Table I. The global B-factors given in the Protein Data Bank files were calculated from TLS tensors and the residual B-factors after refinement with REFMAC (31Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13712) Google Scholar). Model Building—The structure of the catalytic domain of human α-l-fucosidase encompassing Pro6-Trp347 was modeled with the protein homology modeling SWISS-MODEL server using the crystal structure of apo-TM aFuc as template. Given the difficulties in producing an accurate sequence alignment of some particular regions of members of the GH29 family, the accuracy of the resulting model was adjusted manually using the graphics program TURBO-FRODO (29Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Silicon Graphics Corp., 1991: 81Google Scholar). Overall Fold—The crystal structure of TM aFuc was determined at 2.5-Å resolution by the MAD method, taking advantage of the anomalous scattering of selenium, and the model was refined against a native data set at 2.4-Å resolution. Complexes with l-fucose, the reaction product, and fucosyl fluoride, a mechanism-based inhibitor, were obtained by soaking experiments and were refined to 2.8- and 2.25-Å resolution, respectively. Clear unbiased electron density could be observed for both l-fucose and the inhibitor prior to their incorporation in the refinement (Fig. 1, A and B). TM aFuc is a two-domain protein with overall dimensions of 75 × 65 × 55 Å (Fig. 2A). The N-terminal domain (residues 7-359) adopts a (β/α)8-barrel-like fold, with eight parallel β-strands packed around a central axis and surrounded by six α-helices. A depression at the C-terminal ends of the β-strands hosts the active site. The helix corresponding to helix 5 in the classical triose-phosphate isomerase barrel fold is missing in TM aFuc and is replaced by a small loop region, whereas the structural equivalent of helix 6 corresponds to the disordered region Val269-Gly273, which could not be modeled. Despite being located close to the active site, this region does not become structured upon substrate/product binding, as previously observed in related enzyme systems (35Varrot A. Schulein M. Davies G.J. Biochemistry. 1999; 38: 8884-8891Crossref PubMed Scopus (70) Google Scholar, 36Varrot A. Schulein M. Davies G.J. J. Mol. Biol. 2000; 297: 819-828Crossref PubMed Scopus (43) Google Scholar). The core comprising the eight alternating β-strands and six α-helices is decorated by a considerable number of additional α-helices, 310 helices, and extended surface loop regions. The first β-strand is preceded by two small α-helices (residues 11-16 and 21-28) and followed by a long insertion comprising three 310 helices (residues 36-40, 64-66, and 92-94) and three α-helices (residues 68-74, 77-87, and 96-100). A long loop region (residues 128-151) is inserted after β-strand 2 and spatially contiguous to the insertions after β-strand 1. Finally, β-strand 3 is followed by an insertion comprising a 310 helix (residues 188-192). The C-terminal domain of TM aFuc (residues 360-447) is constructed of eight antiparallel β-strands packed into two β-sheets of five and three strands, respectively, forming a two-layer β-sandwich containing a Greek key motif and a small α-helix. The N-terminal domains of the three TM aFuc crystal structures are very similar, with a root mean square deviation for 330 C-α positions in the range of 0.3 Å. Slightly greater differences, ∼0.45 Å for 85 C-α positions, can be observed for the C-terminal domains. Significant differences can be also detected at the level of the relative arrangement of the two domains, indicating that the domain junction is, in fact, a flexible hinge. DALI searches (37Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1268) Google Scholar) performed with the isolated TM aFuc catalytic domain identified several relatives, the top ranked hit being β-N-acetylhexosaminidase (Protein Data Bank code 1hp4) (223 equivalent C-α positions with a root mean square deviation of 3.7 Å). In contrast, the C-terminal domain shares only limited similarity with other proteins, such as the N-terminal domain of Serratia marcescens chitobiase (Protein Data Bank code 1qba) (70 equivalent C-α positions with a root mean square deviation of 2.9 Å) and, with lower scores, with several carbohydrate-binding modules. Whereas the DALI search identified structurally similar modules encountered in a number of glycosidases from various families (GH13, GH15, GH17, GH20, GH27, and GH38), a BLAST search conducted with the C-terminal module alone did not produce any statistically significant hit, not even within GH29 family enzymes. Whether this module has a carbohydrate-binding function in TM aFuc remains unclear, but the lack of significant sequence similarity to other GH29 family enzymes suggests that the C-terminal domain of TM aFuc might have evolved another function. The assignment of a specific function to this domain is rendered particularly difficult by the fact that a number of proteins of diverse function, such as human leukotriene A4 hydrolase (Protein Data Bank code 1hs6), a proteolytic fragment of peptidylprolyl isomerase TLP2 (code 1tul), or the anthrax protective antigen (code 1acc), also fall in the same range of structural similarity. Oligomeric State—In the crystal, TM aFuc assembles into a compact hexameric arrangement, with overall dimensions of 110 × 120 × 140 Å (Fig. 2B). This assembly can be divided into two trimers that stack on each other and are rotated by ∼30°. Upon hexamer formation, the buried surface area (1.6-Å probe radius) per TM aFuc monomer lies between 2380 and 2520 Å2, a value in the highest range compared with other multimeric edifices (38Jones S. Thornton J.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13-20Crossref PubMed Scopus (2239) Google Scholar). The interactions stabilizing the trimers are established exclusively between the catalytic domains, notably between the N-terminal residues, α-helices A3 and A4, and the loop region preceding α-helix A3. The C-terminal domains are projected toward the outer limits of the trimers. The interactions between two trimers involve the N-terminal α-helix, the surface loop regions following β-strands B5 and B6, and a very minor contact area in the C-terminal domain. No covalent bonds are formed between the monomers, even though a cysteine residue, Cys380, is located very close to its counterpart in a neighboring molecule; these two residues are too far apart to form a disulfide bridge. Size exclusion chromatography at low and high ionic strength (100 and 500 mm NaCl, respectively) gave essentially identical elution profiles corresponding to Mr values of ∼300,000, indicating that the hexameric form also prevails in solution. The significance of the hexameric state of TM aFuc is unclear at present. On the one hand, oligomerization is a frequent stabilizing feature of proteins from hyperthermophilic organisms such as T. maritima. On the other hand, it cannot be excluded that oligomerization may have a functional significance in TM aFuc, since most α-l-fucosidases so far characterized, whether from mesophilic or thermophilic organisms, are oligomeric. 3Available at www.brenda.uni-koeln.de. However, the active sites of the single monomers constituting the TM aFuc multimer are well separated, by ∼40 Å, and no interactions between bound substrate and neighboring subunits are detectable. The stability of the hexameric assembly is not disrupted by the introduction of maltose-binding protein at the N terminus. The resulting fusion protein also displays an enzymatic activity comparable with that of native TM aFuc. This suggests that the flexible linker is sufficiently long to allow the accommodation of maltose-binding protein on the outside of the core of the hexameric assembly. Substrate-binding Site—The crystal structures of TM aFuc in complex with l-fucose, the reaction product, allowed us to identify the active site, located in a small pocket formed by the C-terminal ends of the central β-strands of the (β/α)8-domain. The size of the pocket allows a single l-fucose residue to bind, consistent with the exoglycosidase action of α-l-fucosidases. In the product complex, l-fucose is present in the standard 1C4 chair conformation, and its 1-hydroxyl group adopts a β-anomeric configuration, presumably as a result of mutarotation in solution. The sugar is tightly enveloped by the enzyme, and each functional group makes direct contacts with one or more protein side chains, emphasizing a specific substrate recognition pattern for l-fucose (Fig. 1C). The 2-hydroxyl is hydrogen-bonded to the invariant His129 and Trp67 residues. The latter is, in turn, involved in a bidentate hydrogen-bonding network with O-3, together with the conserved side chain of Glu66. The conserved His128 residue stabilizes the axial 4-hydroxyl group, together with the invariant His34 residue, whereas the 1-hydroxyl group is hydrogen-bonded to Asp224. The exocyclic C-6 methyl group is enclosed within a hydrophobic pocket formed by the side chains of the largely conserved residues Phe32, Tyr171, Trp222, and Phe290. No structured water molecules could be observed in the vicinity of the ligand. No major structural rearrangements of the enzyme occur upon ligand binding. The most striking difference can be observed for Phe290, which rotates ∼25° around χ2, and for Tyr64, which flips its side chain by 90° around χ2. This latter movement brings the aromatic ring close to the hydrophobic face of the sugar ring; but instead of establishing a classical “stacking” interaction, often observed in sugar-binding proteins, the plane of the ring is placed perpendicular to the hydrophobic face of the sugar, favoring only very weak van der Waals interactions at a distance of ∼4.5 Å. This situation is often observed at the -1 subsite of glycosidases (subsite nomenclature of Re" @default.
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• W2076261290 date "2004-03-01" @default.
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• W2076261290 title "Crystal Structure of Thermotoga maritima α-l-Fucosidase" @default.
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• W2076261290 doi "https://doi.org/10.1074/jbc.m313783200" @default.
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