Identification of the Catalytic Nucleophile of the Family 29 α-L-Fucosidase from Thermotoga maritima through Trapping of a Covalent Glycosyl-Enzyme Intermediate and Mutagenesis

Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa α-l-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-α-l-fucosyl fluoride. The 104-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an α-l-fucosidase, a key step in the understanding of these important enzymes. Fucose-containing glycoconjugates are key antigenic determinants in many biological processes. A change in expression levels of the enzymes responsible for tailoring these glycoconjugates has been associated with many pathological conditions and it is therefore surprising that little information is known regarding the mechanism of action of these important catabolic enzymes. Thermotoga maritima, a thermophilic bacterium, produces a wide range of carbohydrate-processing enzymes including a 52-kDa α-l-fucosidase that has 38% sequence identity and 56% similarity to human fucosidases. The catalytic nucleophile of this enzyme was identified to be Asp-224 within the peptide sequence 222WNDMGWPEKGKEDL235 using the mechanism-based covalent inactivator 2-deoxy-2-fluoro-α-l-fucosyl fluoride. The 104-fold lower activity (kcat/Km) of the site-directed mutant D224A, and the subsequent rescue of activity upon addition of exogenous nucleophiles, conclusively confirms this assignment. This article presents the first direct identification of the catalytic nucleophile of an α-l-fucosidase, a key step in the understanding of these important enzymes. α-l-Fucosidases (EC 3.2.1.51) are exoglycosidases unique to family 29 in the sequence-based classification of glycoside hydrolases (1Henrissat B. Biochem. J. 1991; 280: 309-316Crossref PubMed Scopus (2624) Google Scholar). 1P. M. Coutinho and B. Henrissat, Carbohydrate-Active Enzymes server: afmb.cnrs-mrs.fr/~cazy/CAZY/index. They are responsible for the removal of l-fucosyl residues from the non-reducing end of glycoconjugates, the most common linkages being α1–2 to galactose and α1–3, α1–4, and α1–6 to N-acetylglucosamine residues. Inactivity of this enzyme leads to the accumulation of fucose-containing glycolipids and glycoproteins in various tissues and results in the clinical condition fucosidosis (3Michalski J.C. Klein A. Biochim. Biophys. Acta. 1999; 1455: 69-84Crossref PubMed Scopus (113) Google Scholar). In humans this autosomal recessive lysosomal storage disease can arise from many different mutations to the FUCA1 gene and, while rare, is a terminal degenerative condition (4Willems P.J. Seo H.C. Coucke P. Tonlorenzi R. O'Brien J.S. Eur. J. Hum. Genet. 1999; 7: 60-67Crossref PubMed Scopus (77) Google Scholar). Fucose-containing oligosaccharides are involved in many key cellular interactions such as inflammatory response (5Springer T.A. Nature. 1990; 346: 425-434Crossref PubMed Scopus (5852) Google Scholar, 6McEver R.P. Moore K.L. Cummings R.D. J. Biol. Chem. 1995; 270: 11025-11028Abstract Full Text Full Text PDF PubMed Scopus (592) Google Scholar, 7Fukuda M. Bioorg. Med. Chem. 1995; 3: 207-215Crossref PubMed Scopus (67) Google Scholar) and antigenic determination (8Feizi T. Trends Biochem. Sci. 1991; 16: 84-86Abstract Full Text PDF PubMed Scopus (127) Google Scholar). Furthermore, changes in fucosylation levels have been observed in many carcinomas (9Hosono J. Narita T. Kimura N. Sato M. Nakashio T. Kasai Y. Nonami T. Nakao A. Takagi H. Kannagi R. J. Surg. Oncol. 1998; 67: 77-84Crossref PubMed Scopus (45) Google Scholar, 10Rapoport E. Le Pendu J. Glycobiology. 1999; 9: 1337-1345Crossref PubMed Scopus (53) Google Scholar). Although many α-l-fucosidases have been identified, their detailed catalytic mechanism and the identities of key active site residues have not yet been elucidated. A complete understanding of the mechanism of action of this class of enzymes is required to enable therapeutic intervention. Recent work has demonstrated that α-l-fucosidases are retaining enzymes; the hydrolyzed hemiacetal product bears the same stereochemical configuration as the original glycoside (11Berteau O. McCort I. Goasdoue N. Tissot B. Daniel R. Glycobiology. 2002; 12: 273-282Crossref PubMed Scopus (70) Google Scholar, 12Cobucci-Ponzano B. Trincone A. Giordano A. Rossi M. Moracci M. J. Biol. Chem. 2003; 278: 14622-14631Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). In the vast majority of these cases this stereochemical outcome arises from a double displacement mechanism within the enzyme active site. This mechanism requires the action of two critical amino acid residues with specific roles for catalysis: one functioning as a general acid/base, the other as a nucleophile. In this class of enzymes these residues are usually found to be carboxylic acids. Glycoside hydrolysis requires the action of the general acid catalyst to assist in the departure of the aglycone oxygen. Simultaneously the other carboxylate residue acts as a nucleophile, attacking the anomeric center of the glycone, generating a covalent glycosyl-enzyme intermediate. Upon diffusion of the aglycone from the active site, this glycosyl-enzyme is subsequently hydrolyzed with general base assistance from the acid/base residue. The hemiacetal thus released retains the same anomeric orientation as the original glycoside because an overall double inversion of configuration has taken place. The identification of these key residues is of fundamental importance to the understanding of the detailed molecular mechanism through which these enzymes operate. General Procedures and Synthesis—All buffer chemicals and other reagents were obtained from the Sigma unless otherwise noted. 2-Deoxy-2-fluoro-α-l-fucosyl fluoride was synthesized by known methodology (13Korytnyk W. Valentekovic-Horvath S. Petrie C.R.I. Tetrahedron. 1982; 38: 2547-2550Crossref Scopus (39) Google Scholar). Cloning, Purification, and Mutagenesis—The coding sequence of the Thermotoga maritima (TM) gene TM0306 was amplified by PCR from genomic DNA (kindly provided by Dr. Wolfgang Liebl) and subcloned into the pDEST17 plasmid (Gateway, Invitrogen) introducing a 15-residue linker and a His6 tag at the N terminus. Protein expression was carried out in Escherichia coli BL21(DE3) pLysS strain and cells were lysed by lysozyme treatment and subsequent freeze-thawing. Protein was purified by Ni2+ affinity and size exclusion chromatographies and sample purity was assessed by SDS-PAGE. The D224A mutant was generated using the QuikChange™ mutagenesis kit (Stratagene), verified by DNA sequencing, but the protein expressed in the conditions described above was insoluble. The coding sequence was therefore subcloned into a derivative of the expression vector pKM596 (14Fox J.D. Routzahn K.M. Bucher M.H. Waugh D.S. FEBS Lett. 2003; 537: 53-57Crossref PubMed Scopus (93) Google Scholar) in which a His6 tag was introduced upstream of the coding sequence of the maltose-binding protein, allowing the expression of the mutant as a His6-MBP 2The abbreviations used are: MBPmaltose-binding proteina → aaxial → axiale → eequatorial → equatorialpNP4-nitrophenolHPLChigh performance liquid chromatography. fusion protein. Expression and purification were carried out as for the native enzyme. The wild type protein was also expressed and purified as a His6-MBP fusion protein for comparison. A detailed description of the cloning, expression, purification, and mutagenesis steps of TM α-l-fucosidase will be described elsewhere. 3G. Sulzenbacher, C. Bignon, T. Nishimura, T. C. Tarling, S. G. Withers, B. Henrissat, and Y. Bourne, manuscript in preparation. maltose-binding protein axial → axial equatorial → equatorial 4-nitrophenol high performance liquid chromatography. Labeling and Proteolysis—Labeling of T. maritima α-l-fucosidase was accomplished by incubating the His6-wild type enzyme (2.43 mg ml–1 final concentration) and 2-deoxy-2-fluoro-α-l-fucosyl fluoride (2.5 mm final concentration) in pH 5.0, 50 mm citrate/phosphate buffer (80 μl total volume) for 1 h at room temperature. After this time digestion buffer (120 μl), pH 2.0, containing pepsin (0.3 mg ml–1) was added. Proteolytic digestion was performed for 1 h and the sample was frozen prior to mass spectrometric analysis. A sample of unlabeled enzyme for comparison was also prepared in the same manner. Electrospray Mass Spectrometric Analysis of the Proteolytic Digest— Mass spectra were recorded on a PE-Sciex API 300 triple-quadrupole mass spectrometer and a PE-Sciex API QSTAR pulsar i (Sciex, Thornhill, Ontario, Canada) equipped with an ionspray ion source. Peptides were separated by reverse-phase HPLC on a LC Packing UltiMate Micro HPLC system (Dionex, Sunnyvale, CA) directly interfaced with the mass spectrometer. In each of the MS experiments the proteolytic digest was loaded onto a C-18 column (LC Packing, 100-Å pepMap, 1 × 150 mm) equilibrated with solvent A (0.05% trifluoroacetic acid, 2% acetonitrile in water). Elution of the peptides was accomplished using a gradient (0–60%) of solvent B over 60 min followed by 85% solvent B over 20 min (solvent B: 0.045% trifluoroacetic acid, 80% acetonitrile in water). Solvents were pumped at a constant flow rate of 50 μl/min. Spectra were recorded in the single quadrupole scan mode or the tandem MS product-ion scan mode. In the single-quadrupole mode the quadrupole mass analyzer was scanned over a mass to charge ratio (m/z) range of 300–2200 Da with a step size of 0.5 Da and a dwell time of 1.5 ms per step. The ion source voltage was set at 5.5 kV, and the orifice energy was 45 V. In the tandem MS daughter-ion scan mode, the spectra were obtained in a separate experiment by selectively introducing the labeled (m/z = 927.5) or unlabeled (m/z = 853) parent ion from the first quadrupole (Q1) into the collision cell (Q2) and observing the product ions in the third quadrupole (Q3). The scan range of Q3 was 50–1100; the step size was 0.5; the dwell time was 1 ms; ion source voltage was 5 kV; orifice energy was 45 V; Q0 = –10; and IQ2 = –48. Metal Ion Dependence—Aliquots (100 μl) of His6-wild type and MBP-wild type α-l-fucosidase were placed into a mini dialysis unit (Pierce) with a molecular weight cut off of 10,000. These units were then dialyzed against a buffer of pH 7.5, 15 mm HEPES containing 10 mm EDTA for 2 h to remove any contaminating divalent metal ions. The dialysis buffer solution was then changed for a fresh solution and dialysis continued for a further 12 h. The dialysis units were then dialyzed against a fresh, pH 7.5, 5 mm HEPES buffer for 2 h to remove residual EDTA. Again the buffer was changed for a fresh solution and dialysis continued for a further 12 h. Enzyme activity of these two proteins was then assayed in the manner described below in the and of divalent metal Enzyme were performed in 50 mm buffer containing mm and the pH range a buffer was buffer was used pH and using the and the His6-wild type enzyme were performed at 60 the enzyme of activity h at this temperature. the were performed at the of A total of μl was used in these pH 5.5 and above a was used to the of the at at pH the product was at were for each pH at 60 by the of and PubMed Scopus Google pH = pH = pH = pH 5.0, = pH = pH = pH = pH = pH = pH = were performed on and The hydrolysis of the fluoride was using a fluoride ion to the of fluoride. This was to a and the using the These were performed at to hydrolysis and were in with a total of each generated the of were the was from to 5 The and were by of the rate to the using Scholar). In cases the observed rate was for methodology was used for the of as a nucleophile. of was performed using 60 pH of the of the for determination of the rate constant not be The pH of the His6-wild type enzyme was therefore by at of 0.5 pH of with were performed at pH in 50 mm citrate/phosphate This from the pH pH was required because of the high of acid of this pH was the of the to pH was at and the rate of product with as rescue were performed at pH in a with in the range of of the D224A fusion protein with were at a of because of the lower of the were to h to for the rate of the was observed to be this time of enzyme Metal Ion metal ion affinity was used in the purification of the His6-wild type an of any metal ion of the enzyme was carried of activity of the His6-wild type or the MBP-wild type enzymes was observed upon dialysis against EDTA. of 10 mm EDTA in the also not the rate of of various divalent metal and at 10 mm not a being observed in A was at high of therefore subsequent were performed in the of mm Analysis and pH for the of by the His6-wild type enzyme were and a is in can be was observed at high these to a in in which arises from the of a to an that is A. Enzyme and of = = and = at pH The and were in to obtained were by to the = = In of this the of in further experiments was of and were at 60 for the hydrolysis of by as a of The enzyme was found to be at pH with This was to enzyme because activity not be upon the pH to The pH of a from the of two carboxylic acid residues in the enzyme active site with a pH of the to a double to of these two residues in the enzyme of and the catalytic nucleophile and acid/base The pH of which the of the two key catalytic residues the step of is also to be a with an at pH The in this are being and the of this is not because of the of the enzyme at of and of wild type T. maritima The the of the to the double in were performed at 60 with as The enzyme was at pH of for any to be fluoride was also as a these being performed at because of the hydrolysis of the fluoride at The catalytic activity of the His6-wild type enzyme with this was to that observed for was observed at The observed for fluoride is most by the of an aglycone to interactions in the a rate was not observed upon of the aglycone with a is of a step for of these = fluoride = that is in the step for these a experiment was The addition of an exogenous nucleophile to the not be to have any upon the rate of the first step of enzyme were this to be the in rate be the rate of be because of the of the glycosyl-enzyme with the exogenous nucleophile with of various of to the a in rate with for and fluoride of the in this a product = with an = and = This product was observed to with an sample of that the product is of this product was not observed in performed in the of or enzyme. is therefore at for of these for hydrolysis of by T. maritima at pH 5.0, × at pH 5.0, × at pH 5.0, not at pH 5.0, × at pH 5.0, × at pH 5.0, at pH 5.0, not at pH 5.0, fluoride. in a to can two energy and the being in solution This with an axial in a to that of enzymes; such a being described as a → a A. Enzyme of Scholar). the be by an → mechanism to The importance of this a → a or → classification is not by the but by the different that be to the catalytic nucleophile. The for the identification of the catalytic nucleophile in retaining has been through the of that a covalent glycosyl-enzyme at a rate that that of the subsequent hydrolysis step. The of this is the accumulation of a and in many cases complete of can then be used to peptides for by liquid to a tandem mass spectrometer and the of the carboxylate residue In the of have R. J. Chem. 1990; Scopus Google Scholar, R. J. Biol. Chem. 1991; Full Text PDF PubMed Google Scholar, J.C. R. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. 2002; PubMed Scopus Google Scholar). This has been found to be with these are observed to be for which the step is J. Biol. Chem. Full Text PDF PubMed Google Scholar, J.D. C. M. PubMed Scopus Google Scholar). In this glycosyl-enzyme this two other of mechanism-based have been These are the C. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google which and the J.D. J. Chem. Scopus Google of which have been used to the catalytic of 2002; PubMed Scopus Google Scholar). The classification of and to the same glycoside family to the and work within this has conclusively demonstrated that and through glycosyl-enzyme S. 2003; PubMed Scopus Google Scholar). In that the catalytic nucleophile of the human was labeled using a fluoride and a fluoride of α-l-fucosidases are in family with other to that is regarding the of the mechanism → a → on the of the with the human S. 2003; PubMed Scopus Google to first and the fluoride as a to the catalytic nucleophile of the T. maritima The of this was known methodology (13Korytnyk W. Valentekovic-Horvath S. Petrie C.R.I. Tetrahedron. 1982; 38: 2547-2550Crossref Scopus (39) Google Scholar). of with 2-Deoxy-2-fluoro-α-l-fucosyl of the enzyme with 2-deoxy-2-fluoro-α-l-fucosyl fluoride not to of this was as a or not in the enzyme active site, of using a fluoride was the rate of fluoride that 2-deoxy-2-fluoro-α-l-fucosyl fluoride was a with = and = This is to for with other for fluoride with the family from S. G. S. FEBS Lett. PubMed Scopus Google Scholar) and observed with fluoride and the family from Biochem. J. PubMed Google Scholar). The of mm over fluoride = is because of the of key interactions with the The is involved in key interactions for and because the of this = for → is to be responsible for the in catalytic Scholar). This is with of the key of the in Chem. PubMed Google Scholar). of the of enzyme or was observed in the of 2-deoxy-2-fluoro-α-l-fucosyl fluoride. this that it is that any over to the activity This has been observed with other with J. 2002; PubMed Scopus Google Scholar). a step for 2-deoxy-2-fluoro-α-l-fucosyl which in accumulation of the that such is the for and also the in because the protein was not to mass spectrometric not for such accumulation was therefore to the peptides of the enzyme with 2-deoxy-2-fluoro-α-l-fucosyl the enzyme was into pH 2 with pepsin was then used to peptide of a size that be by reverse-phase mass of peptides from labeled and native protein identified two peptides that at the same time and and The of the two to that the peptide is with and a of mass as be the with a The ratio of labeled to unlabeled peptide was to be Analysis of peptides by of the enzyme amino acid sequence identified peptides with mass 2 the known of one peptide of this is from pepsin on the sequence of the peptide the and site of was obtained in two separate In the first experiment the peptide of was by and by tandem mass to the B ions (m/z (m/z (m/z and (m/z sequence information for the end of the unlabeled from ions of the unlabeled peptide information the (m/z (m/z (m/z (m/z (m/z (m/z (m/z (m/z (m/z and (m/z This with the mass of the peptide and the sequence of the conclusively confirms the sequence of the labeled peptide to be that This peptide carboxylic acid residues that the A was performed on the labeled peptide to the of The observed was to that in 5 but also a at = not This at = to the peptide (m/z the The of the can therefore be because this peptide one carboxylic acid of the of this peptide with of other of family 29 that this residue is the for this residue being the catalytic nucleophile of the containing the catalytic nucleophile labeled by 2-deoxy-2-fluoro-α-l-fucosyl fluoride in hydrolases of family The α-l-fucosidase are: T. maritima The are in The residue of family 29 α-l-fucosidases is in Analysis of the D224A the importance of this catalytic nucleophile residue a site-directed D224A, was of this mutant as a protein but this was by expression of the mutant as an fusion protein. the of the maltose-binding protein, and for a the α-l-fucosidase was also prepared as an fusion and with the two with the His6-wild type the MBP-wild type fusion protein was found to have a against with the This in is to but not the of this that the of the protein was thus with were performed at a of The D224A mutant was for catalytic activity using and fluoride as in I. This mutant enzyme a activity with as a with wild type enzyme. was observed at 1 mm therefore a of be not be because of the of The 104-fold in observed in this is but that found with nucleophile of a is common M. M. R. M. C. E. A. PubMed Scopus Google Scholar, J. Chem. Scopus Google Scholar, Biochem. 1999; PubMed Scopus Google Scholar). to the × observed for the nucleophile mutant of B. J. Biol. Chem. 1995; 270: Full Text Full Text PDF PubMed Scopus Google Scholar). The residual catalytic activity of this mutant is to arise from wild type because in that a on with a to that of the wild type enzyme be this mutant was assayed with because of the of the fluoride and the hydrolysis activity of this enzyme above hydrolysis be using fluoride as a at The activity observed with this mutant enzyme is with the of this residue as catalytic nucleophile. of D224A with as of this mutant was performed to catalytic activity be by the addition of exogenous is that the to of catalytic residues in active in which can Chem. Biol. PubMed Scopus Google Scholar). In cases these can then to the enzyme by in of the residue. In the of the of activity can wild type levels J. Chem. Scopus Google Scholar, PubMed Scopus Google Scholar). The on on the other is with a being common R. 2002; PubMed Scopus Google Scholar). and were for their to to this mutant using as a The addition of a of mm) was to the rate of by to The rate was to be on to was observed and further rate was were also obtained with a rate being observed in this This most the of the exogenous in the enzyme upon removal of the catalytic nucleophile. and are observed to with with of and 5 a of active site and mutagenesis have conclusively identified Asp-224 to be the catalytic nucleophile of the T. maritima This the first direct identification of the catalytic nucleophile for any family 29 The of this α-l-fucosidase with a in to the by this class of inactivator with a → a that are of this enzyme in common with a similarity also observed with human S. 2003; PubMed Scopus Google Scholar). Coutinho is for with the sequence

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