A New Sialidase Mechanism
2009; Elsevier BV; Volume: 284; Issue: 26 Linguagem: Inglês
10.1074/jbc.m109.003970
ISSN1083-351X
AutoresThomas Morley, Lisa M. Willis, Chris Whitfield, Warren W. Wakarchuk, Stephen G. Withers,
Tópico(s)Genomics and Phylogenetic Studies
ResumoBacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endo-sialidases. This family is unusual because all other previously reported sialidases outside of this family are exo- or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a series of pH values revealed a dependence on a single protonated group of pKa 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases. Bacteriophages specific for Escherichia coli K1 express a tailspike protein that degrades the polysialic acid coat of E. coli K1 that is essential for bacteriophage infection. This enzyme is specific for polysialic acid and is a member of a family of endo-sialidases. This family is unusual because all other previously reported sialidases outside of this family are exo- or trans-sialidases. The recently determined structure of an endo-sialidase derived from bacteriophage K1F (endoNF) revealed an active site that lacks a number of the residues that are conserved in other sialidases, implying a new, endo-sialidase-specific catalytic mechanism. Using synthetic trifluoromethylumbelliferyl oligosialoside substrates, kinetic parameters for hydrolysis at a single cleavage site were determined. Measurement of kcat/Km at a series of pH values revealed a dependence on a single protonated group of pKa 5. Mutation of a putative active site acidic residue, E581A, resulted in complete loss of sialidase activity. Direct 1H NMR analysis of the hydrolysis of trifluoromethylumbelliferyl sialotrioside revealed that endoNF is an inverting sialidase. All other wild type sialidases previously reported are retaining glycosidases, implying a new mechanism of sialidase action specific to this family of endo-sialidases. The important role played by sialylated glycoconjugates (1Schauer R. 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In this paper we describe the development of substrates for the convenient assay of endo-sialidases and use these reagents to establish that the endo-sialidase follows a completely different chemical mechanism to all other sialidases, resulting in inversion of anomeric configuration. polysialic acid α-linked trifluoromethylumbelliferyl glycoside a sialic acid polymer of length n with α-linked TFMU aglycone high pressure liquid chromatography 3-(cyclohexylamino)-1-propanesulfonic acid. polysialic acid α-linked trifluoromethylumbelliferyl glycoside a sialic acid polymer of length n with α-linked TFMU aglycone high pressure liquid chromatography 3-(cyclohexylamino)-1-propanesulfonic acid. All of the chemicals were of analytical grade purchased from Sigma-Aldrich, unless otherwise stated. Trifluoromethylumbelliferyl α-sialoside was prepared as previously described (54Engstler M. Talhouk J.W. Smith R.E. Schauer R. Anal. Biochem. 1997; 250: 176-180Crossref PubMed Scopus (19) Google Scholar). Sialyltransferase from Campylobacter jejuni (Cst-II) (55Chiu C.P. Watts A.G. Lairson L.L. Gilbert M. Lim D. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Mol. Biol. 2004; 11: 163-170Crossref PubMed Scopus (177) Google Scholar) and trans-sialidase from Trypanosoma cruzi (56Damager I. Buchini S. Amaya M.F. Buschiazzo A. Alzari P. Frasch A.C. Watts A. Withers S.G. Biochemistry. 2008; 47: 3507-3512Crossref PubMed Scopus (54) Google Scholar) were expressed and purified as previously described. CMP-N-acetyl neuraminic acid was a kind gift from Neose Technologies Inc. All DNA isolations, restriction enzyme digestions, ligations, and transformations were performed as recommended by the supplier. All other enzymes were obtained from New England Biolabs and Sigma-Aldrich. A buffered (50 mm HEPES, pH 7.5) solution (422 μl total) of trifluoromethylumbelliferyl α-sialoside (10.8 mg, 21.1 μmol) containing manganese chloride (10 mm) was incubated at room temperature with CMP-N-acetyl neuraminic acid (67 mg, 105 μmol) in the presence of Cst-II (2.8 mg/ml, 200 μl) and alkaline phosphatase (130 units/μl, 2 μl) for 2 h. After centrifugation (10,000 × g, 2 min), the supernatant was filtered (0.44-μm filter; Millipore), applied to a Biogel-P4 size exclusion column (25 mm × 90 mm) at 4 °C, and eluted with water (7 ml/h). Product-containing fractions were identified by UV (280 nm) and TLC analysis (ethyl acetate/methanol/water/acetic acid, 4:2:1:0.1 ratio mobile phase), pooled, and lyophilized. The mixture of oligomers obtained was subjected to C18 reverse phase silica HPLC (Phenomenex Luna 15-μm C18(2) 250 × 21.2 mm column, Waters 600 multi-solvent delivery system, and Waters 2487 multi-channel UV-detection system). Oligosaccharides from Sia5-TFMU to Sia3-TFMU were eluted in water, increasing to 20% acetonitrile to elute the shorter oligomers. Product-containing fractions were located by absorbance at 324 nm and after being confirmed by TLC analysis were pooled and lyophilized. Genomic DNA was isolated from E. coli bacteriophage K1F (57Petter J.G. Vimr E.R. J. Bacteriol. 1993; 175: 4354-4363Crossref PubMed Google Scholar) by incubating phage with proteinase K (25 μg/ml) in 50 mm Tris-HCl, 75 mm NaCl, 6.25 mm EDTA, pH 7.4, 1% SDS at 55 °C for 1 h followed by extraction in 1:1 phenol:chloroform and ethanol precipitation. The endo-sialidase gene was amplified from genomic DNA in a two-step pull-through PCR procedure to remove an internal NdeI site. In this amplification strategy, the gene was also truncated 735 bp from the 5′-end to make it the same length as the construct from which the crystal structure was generated (15Stummeyer K. Dickmanns A. Mühlenhoff M. Gerardy-Schahn R. Ficner R. Nat. Struct. Mol. Biol. 2005; 12: 90-96Crossref PubMed Scopus (158) Google Scholar). The resulting gene is then 2463 base pairs long and encodes a protein corresponding to amino acids 246–1065 of the full-length K1F protein. To reduce confusion that would arise from different numbering of the residues, we will base our numbering on the full-length protein so that Glu581 in our truncated protein corresponds to Glu581 in the full-length protein. Primers for the endo-sialidase were as follows: 5′-GGCGGACATATGGCTAAAGGGGATGGTGTCACTG (external forward), 5′-GGCGGAGTCGACCTATTACTTCTGTTCAAGAGCAGAAAGTC (external reverse), 5′-CCATTTTAACCCATGGACTTATGGAGATAACTCAGCG (internal reverse), and 5′-CGCTGAGTTATCTCCATAAGTCCATGGGTTAAAATGG (internal forward). PCR was performed using Phusion polymerase and the program: 94 °C for 5 min, 30 cycles of 94 °C for 30 s, 55 °C for 30 s and 72 °C for 60 s, and finally 72 °C for 10 min. DNA was purified using phenol-Tris extraction and ethanol precipitation. Genes digested with NdeI and SalI were ligated into pCWmalE-thrombin (58Willis L.M. Gilbert M. Karwaski M.F. Blanchard M.C. Wakarchuk W.W. Glycobiology. 2008; 18: 177-186Crossref PubMed Scopus (41) Google Scholar) and then used to transform E. coli AD202 (CGSC 7297) by electroporation. The endo-sialidase E581A mutant was made using the same pull-through PCR method described above using the following primers: 5′-GAGTATGAACCAGATGCGTCAGCGCCGTGCATCAAGTACTATG-3′ (forward) and 5′-CATAGTACTTGATGCACGGCGCTGACGCATCTGGTTCATACTC-3′ (reverse). Plasmids were isolated using High Pure Plasmid Isolation kit (Sigma-Aldrich). DNA sequencing was performed using an Applied Biosystems model 3100 automated DNA sequencer and the manufacturer's cycle sequencing kit at the Guelph Molecular Supercenter. Recombinant E. coli strains were grown in LB broth containing ampicillin (150 μg/ml) at 37 °C for 2 h. Gene expression was induced with 0.5 mm isopropyl 1-thio-β-d-galactopyranoside and grown at 30 °C for 24 h. The cells overexpressing MalE-EndoNF were resuspended in 20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 2 mm EDTA and lysed by French Press in the presence of a protease inhibitor mixture pellet (Roche Applied Science). The lysate was centrifuged at 27000 × g for 30 min at 4 °C to remove cell debris. The supernatant was treated with DNaseI (20 μg/ml) and RNase A (10 μg/ml) in 10 mm MgCl2 on ice for 30 min and then centrifuged at 100,000 × g for 60 min at 10 °C. The supernatant was applied to amylose resin (New England Biolabs) equilibrated in 20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 2 mm EDTA. MalE-EndoNF was eluted with 10 mm maltose in 20 mm Tris-HCl, pH 7.5, 200 mm NaCl, 2 mm EDTA, and the fractions were analyzed by SDS-PAGE. Fractions with significant amounts of MalE-EndoNF were pooled. Expression and purification of the endo-sialidase E581A mutant was carried out under identical conditions. A buffered (20 mm phthalate, 50 mm NaCl, pH 4.5) solution (20 μl total) of the oligomer (2 mm in reaction) was incubated with endoNF (2.6 mg/ml, 4 μl). Aliquots were taken over a period of 30 min and subjected to TLC analysis (regular phase silica with an ethyl acetate/methanol/water/acetic acid, 4:2:1:0.1 ratio mobile phase) along with TLC standards of each oligomer and a control carried out in the absence of enzyme. Kinetic analyses were performed at pH 4.5 (20 mm sodium phosphate/citrate buffer, 50 mm NaCl), monitoring the release of the free coumarin at 380 nm (ϵ = 1580 m−1 cm−1) using a Varian Cary-4000 UV-visible spectrophotometer. The data were analyzed using GraFit software from Erithacus Software. Enzyme stability assays were conducted by preincubation of aliquots of EndoNF with the desired buffer (20 mm with 50 mm NaCl) for 30 min at 37 °C before being assayed (at pH 4.5) and compared against a standard incubated at pH 7.5 (20 mm Tris-HCl, 50 mm NaCl) for the same period. Measurements of kcat/Km values at a series of pH values were carried out by the substrate depletion method (59Joshi M.D. Sidhu G. Pot I. Brayer G.D. Withers S.G. McIntosh L.P. J. Mol. Biol. 2000; 299: 255-279Crossref PubMed Scopus (190) Google Scholar) using citrate/phosphate buffer (20 mm, pH 2.2–6.5, 50 mm NaCl) and phosphate (20 mm, pH 6.0–8.0, 50 mm NaCl). Kinetic analyses were performed at pH 4.5 (20 mm sodium phosphate/citrate buffer, 50 mm NaCl), using a stopped assay. The reaction was initiated by the addition of endoNF (0.5 mg/ml, 5 μl) to a buffered solution (total volume, 25 μl) of Sia3-TFMU substrate. Aliquots of the mixture (5 μl) were taken at 0, 30, 90, and 120 s and diluted into 50 mm CAPS pH 10.0 buffer (995 μl). The fluorescence of the released coumarin at each time point was measured (λexit = 385 nm, λemit = 502 nm) using a Varian Cary Eclipse fluorescence spectrophotometer and plotted to give an initial reaction rate at each concentration. The data were analyzed using Grafit software from Erithacus Software. Aliquots of a solution of Sia3-TFMU (4.0 mm) were added to each of two fluorescence cuvettes: one containing mutant EndoNF (200 μl, 5 mg/ml) in 20 mm sodium phosphate/citrate, 50 mm NaCl pH 4.5 buffer (total volume, 800 μl) and the second containing only buffer. After the addition of each aliquot of substrate followed by thorough mixing and thermal equilibration to 25 °C, the difference in fluorescence (λexit = 328 nm, λemit = 495 nm) between the two cuvettes was measured using a Varian Cary Eclipse fluorescence spectrophotometer. After correcting for differences in volume and enzyme fluorescence, a plot of the difference in fluorescence versus concentration was analyzed using GraFit to determine the maximum difference in fluorescence. The data were then analyzed using a Scatchard plot (60Jolley M.E. Glaudemans C.P. Carbohydr. Res. 1974; 33: 377-382Crossref PubMed Scopus (64) Google Scholar) to determine Kd, and the experiment was repeated twice to determine accuracy. Sia3-TFMU was dissolved in deuterated buffer (10 mm phthalate pH 4.5, 25 mm NaCl) to give a final substrate concentration of 1.3 mm. 1H NMR spectra were obtained on a Bruker Avance 400inv spectrometer fitted with 5 mm BBI-Z probe. The data were collected at 298 K over 16 scans at 2-min intervals after the addition of a solution of endoNF in deuterated buffer (40 μl, 5.8 mg/ml in 20 mm Tris-HCl, pH 7.5). NMR experiments were performed using a water suppression protocol by irradiating with a low power (55 dB) continuous wave pulse centered at 4.7 ppm during the relaxation period (d1 = 2 s). Acquisition was performed following a delay (20 μs) after a 90-degree pulse (9.5 μs at 1 dB) 4 μs after the relaxation period. The data were analyzed using ACD software. The mechanistic and kinetic study of endoNF required a suitable homogeneous substrate with a defined cleavage site that can be easily monitored. Excellent candidates for such substrates would be aryl-oligosaccharides that are cleaved exclusively at the aryl glycoside bond because liberation of the phenol(ate) can be monitored directly by UV-visible or fluorescence spectroscopy. This outcome can be favored if a phenol of relatively low pKa is used, because not only is the rate of cleavage of the aryl glycoside likely to be greater than that of inter-sugar bonds, but also the phenol is likely to be released in a detectable phenolate form at ambient pH. Such a strategy has proved valuable with other endo-glycosidases such as amylases (61Henkel E. Sollmann C. Henkel R. Clin. Chem. 1983; 29 (1182): 1182Google Scholar, 62Brayer G.D. Sidhu G. Maurus R. Rydberg E.H. Braun C. Wang Y. Nguyen N.T. Overall C.M. Withers S.G. Biochemistry. 2000; 39: 4778-4791Crossref PubMed Scopus (195) Google Scholar) and cellulases (63Tull D. Withers S.G. Biochemistry. 1994; 33: 6363-6370Crossref PubMed Scopus (112) Google Scholar). In this case a TFMU leaving group was chosen, because its sialoside is relatively stable to spontaneous hydrolysis, and cleavage can be monitored by both UV absorption and fluorescence emission (54Engstler M. Talhouk J.W. Smith R.E. Schauer R. Anal. Biochem. 1997; 250: 176-180Crossref PubMed Scopus (19) Google Scholar) spectroscopy. Previous studies indicated that the minimum substrate length required by endoNF is an oligosialic acid with a degree of polymerization of 5 (i.e. Sia5) (53Hallenbeck P.C. Vimr E.R. Yu F. Bassler B. Troy F.A. J. Biol. Chem. 1987; 262: 3553-3561Abstract Full Text PDF PubMed Google Scholar); thus a range of oligomeric TFMU sialosides was synthesized to find the optimal aryl oligosialoside for kinetic analysis. This was achieved by oligosialylation of a chemically synthesized TFMU sialoside acceptor using an oligosialytransferase from C. jejuni (Cst-II), which catalyzes the formation of α-2,8-linked sialic acid oligomers (55Chiu C.P. Watts A.G. Lairson L.L. Gilbert M. Lim D. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Mol. Biol. 2004; 11: 163-170Crossref PubMed Scopus (177) Google Scholar). Use of five equivalents of the donor CMP-sialic acid (Fig. 1) led to the desired range of oligomers. The oligomeric mixture was separated by size exclusion chromatography, followed by C18 reverse phase silica HPLC. The degradation of each of the synthetic oligomers by endoNF was monitored by TLC analysis, revealing that endoNF does not hydrolyze the monomeric (Sia-TFMU) or dimeric (Sia2-TFMU) sialosides significantly over a 2-h period. However, the trimeric (Sia3-TFMU) sialoside is degraded with exclusive cleavage at the coumarin-sialoside bond. The tetramer (Sia4-TFMU) was also hydrolyzed, with formation of two TFMU-containing species, trifluoromethylumbelliferone and the TFMU sialoside monomer, indicating multiple cleavage sites (Fig. 2). The pentamer (Sia5-TFMU) could not be purified to homogeneity and was not tested as a substrate. Based upon these data, it appears that endoNF has at least three subsites on the nonreducing side of the cleavage site, and the minimum substrate requirement is for the −1, −2, and −3 subsites (64Davies G.J. Wilson K.S. Henrissat B. Biochem. J. 1997; 321: 557-559Crossref PubMed Scopus (839) Google Scholar) to all be occupied. This observation is consistent with the cleavage patterns of other endo-sialidases that have been reported using simple oligosialoside substrates (53Hallenbeck P.C. Vimr E.R. Yu F. Bassler B. Troy F.A. J. Biol. Chem. 1987; 262: 3553-3561Abstract Full Text PDF PubMed Google Scholar, 65Kataoka Y. Miyake K. Iijima S. J. Biosci. Bioeng. 2006; 101: 198-201Crossref PubMed Scopus (5) Google Scholar, 66Pelkonen S. Pelkonen J. Finne J. J. Virol. 1989; 63: 4409-4416Crossref PubMed Google Scholar) and particularly with a very recent publication showing that the tetramer is the true minimum substrate (67Schwarzer D. Stummeyer K. Haselhorst T. Freiberger F. Rode B. Grove M. Scheper T. von Itzstein M. Mühlenhoff M. Gerardy-Schahn R. J. Biol. Chem. 2009; 284: 9465-9474Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Our data also serve to confirm that the enzyme does not work via an exo-sialidase mode from the nonreducing terminus but rather via an endo-mode. Because the trimer (Sia3-TFMU) is cleaved exclusively at the coumarin-sialoside bond, it was chosen as the substrate for further kinetic and mechanistic analysis by monitoring the release of free trifluoromethylumbelliferyl. An initial pH versus rate profile suggested that the pH optimum was at ∼4.5 (data not shown) and kinetic parameters of kcat of 77 ± 5 min−1 and a Km of 0.68 ± 0.09 mm were obtained for hydrolysis of TFMU sialotrioside at this pH. These values are similar to those obtained for other sialic acid oligomers (e.g. Km = ∼1.2 mm and kcat = 72 min−1 for a degree of polymerization 10) (53Hallenbeck P.C. Vimr E.R. Yu F. Bassler B. Troy F.A. J. Biol. Chem. 1987; 262: 3553-3561Abstract Full Text PDF PubMed Google Scholar) and closely correlate with the recent analysis of the degradation of a sialic acid tetramer by endoNF (67Schwarzer D. Stummeyer K. Haselhorst T. Freiberger F. Rode B. Grove M. Scheper T. von Itzstein M. Mühlenhoff M. Gerardy-Schahn R. J. Biol. Chem. 2009; 284: 9465-9474Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Analysis of the enzyme stability at a series of pH values (Fig. 3) showed that endoNF is unstable below pH 4 and above pH 12 over the time period tested, thus further studies were carried out within this range. A more detailed pH dependence was carried out by measuring values of kcat/Km at each pH, using the substrate depletion method (59Joshi M.D. Sid
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