The Structure of Clostridium perfringens NanI Sialidase and Its Catalytic Intermediates

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97Å resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97Å resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5Å resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2Å resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested. Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97Å resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97Å resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5Å resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2Å resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested. Clostridium perfringens is a Gram-positive anaerobic bacterium that causes life-threatening gas gangrene and enterotoxemia in humans. C. perfringens infections are characterized by the release of large amounts of toxins and enzymes that can cause massive destruction of the host tissue, putting the organism into the category of flesh-eating microbes (1Rood J.I. Annu. Rev. Microbiol. 1998; 52: 333-360Crossref PubMed Scopus (275) Google Scholar). Exo-sialidases are among the virulence factors, two of which (nanH and nanI) had been characterized prior to the sequencing of the complete C. perfringens genome, which revealed a third, nanJ (2Shimizu T. Ohtani K. Hirakawa H. Ohshima K. Yamashita A. Shiba T. Ogasawara N. Hattori M. Kuhara S. Hayashi H. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 996-1001Crossref PubMed Scopus (591) Google Scholar). The nanH gene product is a 43-kDa sialidase (3Kruse S. Kleineidam R.G. Roggentin P. Schauer R. Protein Expression Purif. 1996; 7: 415-422Crossref PubMed Scopus (18) Google Scholar) that is not secreted, whereas the nanI gene product is a 77-kDa sialidase (4Traving C. Schauer R. Roggentin P. Glycoconj. J. 1994; 11: 141-151Crossref PubMed Scopus (31) Google Scholar) that is secreted. These two sialidases have been extensively characterized and shown to exhibit very different kinetic and biochemical properties (5Roggentin P. Kleineidam R.G. Schauer R. Biol. Chem. Hoppe-Seyler. 1995; 376: 569-575Crossref PubMed Scopus (40) Google Scholar). The nanJ gene product has yet to be characterized but is predicted to form a 129-kDa sialidase as it contains the conserved catalytic and “bacterial neuraminidase repeat” signatures of a sialidase (6Roggentin P. Kleineidam R.G. Schauer R. Glycoconj. J. 1989; 6: 349-353Crossref PubMed Scopus (157) Google Scholar). Sialidases, or neuraminidases, catalyze the removal of terminal sialic acids from a variety of glycoconjugates and play an important role in pathogenesis, bacterial nutrition, and cellular interactions. Crystal structures of a growing number of exo-sialidases are available from bacteria (7Crennell S.J. Garman E.F. Laver W.G. Vimr E.R. Taylor G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9852-9856Crossref PubMed Scopus (236) Google Scholar, 8Crennell S.J. Garman E. Laver G. Vimr E.R. Taylor G. Structure (Camb.). 1994; 2: 535-544Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 9Gaskell A. Crennell S.J. Taylor G. Structure (Camb.). 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar), viruses (10Varghese J.N. Laver W.G. Colman P.M. Nature. 1983; 303: 35-40Crossref PubMed Scopus (732) Google Scholar, 11Burmeister W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar, 12Crennell S.J. Takimoto T. Portner A. Taylor G. Nat. Struct. Biol. 2000; 7: 1068-1074Crossref PubMed Scopus (350) Google Scholar), trypanosomes (13Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar, 14Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar), leech (15Lou Y. Li S.-C. Chou M.-Y. Li Y.-T. Lou M. Structure (Camb.). 1998; 6: 521-530Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and man (16Chavas L.M. Tringali C. Fusi P. Venerando B. Tettamanti G. Kato R. Monti E. Wakatsuki S. J. Biol. Chem. 2005; 280: 469-475Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). All sialidases share the same six-bladed β-propeller fold for their catalytic domains, with conservation of key catalytic amino acids (17Taylor G. Curr. Opin. Struct. Biol. 1996; 6: 830-837Crossref PubMed Scopus (212) Google Scholar). The nonviral sialidases also have conserved bacterial neuraminidase repeats or Asp boxes ((S/T)XD(X)GXT(W/F)) occurring between one and five times along the sequence. The bacterial neuraminidase repeats occur at topologically identical positions in the β-propeller fold, remote from the active site, but any function beyond dictating a structural fold is unknown. Many sialidases possess domains in addition to the catalytic domain, placed upstream, downstream, or even inserted within the β-propeller domain; the Vibrio cholerae sialidase has two lectin domains flanking the catalytic domain, one of which binds sialic acid (18Moustafa I. Connaris H. Taylor M. Zaitsev V. Wilson J.C. Kiefel M.J. von Itzstein M. Taylor G. J. Biol. Chem. 2004; 279: 40819-40826Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar); Micromonospora viridifaciens sialidase has a galactose-binding domain C-terminal to the catalytic domain and is positioned above the active site (20Newstead S.L. Watson J.N. Bennet A.J. Taylor G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2005; 61: 1483-1491Crossref PubMed Scopus (43) Google Scholar); the leech sialidase has a lectin-like domain N-terminal to the catalytic domain (15Lou Y. Li S.-C. Chou M.-Y. Li Y.-T. Lou M. Structure (Camb.). 1998; 6: 521-530Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and the trypanosome (trans-) sialidases have a lectin-like domain C-terminal to the catalytic domain (13Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar, 14Buschiazzo A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). It has been suggested that the presence of these carbohydrate-binding modules increases the catalytic efficiency of the sialidases, particularly in the presence of polysaccharide substrates (21Thobhani S. Ember B. Siriwardena A. Boons G.-J. J. Am. Chem. Soc. 2002; 125: 7154-7155Crossref Scopus (63) Google Scholar). Many glycoside hydrolases have additional carbohydrate-binding modules, particularly those involved in the degradation of insoluble polysaccharides such as cellulose and starch, and these carbohydrate-binding modules show a great diversity in ligand recognition and folds (22Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). Sialidases hydrolyze sialic acids from glycoconjugates with retention of configuration at the anomeric center (23Chong A.K. Pegg M.S. Taylor N.R. von Itzstein M. Eur. J. Biochem. 1992; 207: 335-343Crossref PubMed Scopus (190) Google Scholar). The early mechanistic and structural studies on the influenza virus sialidase led to the proposal that the mechanism involved distortion of the sialic acid toward an oxocarbenium ion transition state. Structures of the influenza sialidase complexed with the inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en), 4The abbreviations used are: Neu5AcN-acetylneuramic acidNeu5Ac2en2-deoxy-2,3-dehydro-N-acetylneuraminic acidPDBProtein Data BankPEGpolyethylene glycolr.m.s.root mean square. a putative transition-state analogue, led to the theory that the positive charge on the transition state be by the conserved the of this W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar). complexes of bacterial and trypanosomal sialidases have this with the conserved to the of with a mean of and (13Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar, G. Crennell S.J. C. M. Y. and of Scholar). The mechanism was one in which a conserved acid as a acid the of the between the sialic acid and the The oxocarbenium transition state a similar to that in sialidases, a as the catalytic the positive charge on the and is by catalysis from the conserved which its W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar, A.K. Pegg M.S. Taylor N.R. von Itzstein M. Eur. J. Biochem. 1992; 207: 335-343Crossref PubMed Scopus (190) Google Scholar). N-acetylneuramic acid 2-deoxy-2,3-dehydro-N-acetylneuraminic acid Protein Data mean square. This mechanism is different from that for other retaining which have been shown to via a covalent the of a and an Curr. Opin. Chem. Biol. 2000; PubMed Scopus Google Scholar). The structures of sialidases that was positioned to form a covalent intermediate It was this that of the to an covalent of the intermediate W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar, A.K. Pegg M.S. Taylor N.R. von Itzstein M. Eur. J. Biochem. 1992; 207: 335-343Crossref PubMed Scopus (190) Google Scholar, Y. Li Li Lou M. J. Mol. Biol. 1998; Scholar). Recent kinetic on the using a acid led to the of a covalent intermediate on the conserved I. Amaya M.F. A. Alzari P.M. Frasch A.C. J. Am. Chem. Soc. 125: Scopus Google Scholar). This led to the proposal that the conserved catalytic was in the for this of glycosidases. This kinetic was further by an structural of the T. snapshots of the the covalent intermediate M.F. I. A. T. A. Paris G. Frasch A.C. Alzari P.M. Structure (Camb.). 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and the with A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). It is that the of a as the a amino is a of the that occur with the of sialic acids I. Amaya M.F. A. Alzari P.M. Frasch A.C. J. Am. Chem. Soc. 125: Scopus Google Scholar). is that is a to for the of with other J.N. V. Bennet A.J. PubMed Scopus Google Scholar). The structural and kinetic of covalent with the sialidase further to the that all exo-sialidases a similar mechanism the of a P. Alzari P.M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). this we the structure of the catalytic domain of the C. perfringens NanI sialidase in its ligand form and in with acid structure at resolution. the was the of with structure at of the inhibitor by the an by with the transition-state analogue at 1.5Å resolution was at a covalent intermediate at resolution was by NanI with acid structure These structures the role by the active site and the suggestion that catalysis by sialidases via a very similar mechanism to that of other retaining glycosidases. Data catalytic domain of the C. perfringens NanI sialidase was and as S. Taylor M. Taylor G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). All on at the at and at a of for the ligand and beyond but for of to The of NanI with was from a crystal for in in was by at into a in The ligand and to the with one in the The covalent was by a crystal in acid in for prior to and This in a of the to the with similar but with two in the All by for a to a and a in All with using and to structure with all of which are of the Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google and in to the resolution and and and of of in a Structure was using P. J. M. L.M. T. G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; PubMed Scopus Google Scholar) with a of the sialidase from the leech as a S. Taylor M. Taylor G. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; PubMed Scopus Google Scholar). The leech sialidase with NanI and within the catalytic The of the β-propeller domain of the leech sialidase the domain that is inserted between the and of the of the was used to the using all to A. R. Nat. Struct. Biol. 6: PubMed Scopus Google Scholar) was used to the of the and to in the amino acids using to This of a amino and with a The and and and the and and PubMed Scopus Google Scholar) was to the at resolution of the structure from the of between of with the M. Acta Sect. A. PubMed Scopus Google Scholar). with to one of and one of with PubMed Scopus Google Scholar) and to by two of for two to a of in and was by a of and further of in which a further in The of was used to the or of the atomic positions using The not but in the active The ligand structure was in a similar with using the structure as the The and covalent intermediate using from the Crystallogr. Sect. D Biol. Crystallogr. 1994; PubMed Scopus Google Scholar). The covalent between and was as a of P. Alzari P.M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of the covalent led to a of the covalent to and at the and the suggested the of and the covalent intermediate at in and a covalent of of the structures from PubMed Scopus Google Scholar), and for atomic are in of to form was using of a of for as the NanI of NanI of in and of in and a of in and of in on a with a at K. with and a of for the addition of NanI to the of the was by of the the structure of the catalytic domain of NanI is to a resolution of in its ligand The NanI structure folds into two domains as a six-bladed β-propeller catalytic sialidase domain formed by and and a domain formed by and The between NanI and the leech sialidase with which NanI is for positions The domain in which was from the a structural to the domain in the leech This domain is in amino acids with in the leech sialidase, with a of and is an The for positions is The on the of NanI a very of bacterial sialidases, with the remote from the active site a charge that may to the toward its substrates and The contains two within the β-propeller fold by and of the is in a very similar to a key site in the sialidase from a sialidase S.J. Garman E. Laver G. Vimr E.R. Taylor G. Structure (Camb.). 1994; 2: 535-544Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). The in NanI not to function in the same in that play role in active site the from the complexes of NanI with and acid. the of the of with the active site, and an of the active of the ligand structure and all three ligand are a number of between the active of and sialidases, a number of key have been their (17Taylor G. Curr. Opin. Struct. Biol. 1996; 6: 830-837Crossref PubMed Scopus (212) Google Scholar) and are shown in These a and that with the of The of the is by a conserved acid and a acid with other and and to the of the conserved of sialidase active is the All sialidase active have a to the of the but the that form this are not the NanI this is of and that a on the in an to a in the T. active of the NanI active with the as of the three ligand complexes and ligand with with and the covalent fluorinated ligand with All of the between in the active site and to all three are conserved in the different complexes as are three and in between the complexes as to the ligand not with the but the of three as that with the that with the and that a key role in the the ligand and to the of and ligand intermediate in a resolution structure of the that it is in its with the a The of a with the and with a also by The of with and that are conserved among bacterial and The in a with the of the also with an that has been in complexes of bacterial sialidases with The a to a above the The a of five The a to the also with the and two to and a The terminal two one to the same by and the anomeric is the of the catalytic which is for on the anomeric at a of The of a with the of resolution structure that a The between the of and of the ligand to with in the by with the to of the and a with the resolution structure acid to in an as in the T. M.F. I. A. T. A. Paris G. Frasch A.C. Alzari P.M. Structure (Camb.). 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) and T. sialidase P. Alzari P.M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) covalent that is a of and to for the with the and with the It to of the ligand and to the are the of with two in the The of the the into the and this to two of these of of to form at was as a using of NanI and revealed that the of the of at a of by with the in of the of was the of a in the of the of the of and the of an additional at as shown in The of the at is with in the in in a in the of The of is of of the with the identical but in the of NanI in of the of the of the at or of these that NanI sialidase can catalyze the of to The of from has been for the influenza virus neuraminidase W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar) and the V. cholerae sialidase (18Moustafa I. Connaris H. Taylor M. Zaitsev V. Wilson J.C. Kiefel M.J. von Itzstein M. Taylor G. J. Biol. Chem. 2004; 279: 40819-40826Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). It has also been that sialidase can catalyze the of by of of M. J. Biochem. 1983; PubMed Scopus Google Scholar). The structures provide high resolution snapshots of the catalytic of the NanI The the in a and that is of the similar sialic acid was in the between an acid of T. and M.F. I. A. T. A. Paris G. Frasch A.C. Alzari P.M. Structure (Camb.). 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This of the in a of the it within of the acid for The transition by the with the to a the between and the of the from to of the covalent intermediate and the release of the further in the of the The is to into the with a to the catalytic This in the the in of and of the substrate that is to form the covalent with the anomeric from its in the as as the of by with the covalent is a in the of one of the of the by its from the to from in the The atomic resolution of the very of from which can be This has been used to to key catalytic in S.J. S. J. Davies G.J. J. Chem. Soc. Chem. 2004; Scopus Google Scholar). the the of of are and that the charge on this is and that the is not all sialidases to the of this conserved acid a with the of the catalytic The T. suggested that a acid as a for the of the on the anomeric in a charge M.F. I. A. T. A. Paris G. Frasch A.C. Alzari P.M. Structure (Camb.). 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). This has also support from a on of the bacterial sialidase from M. viridifaciens J.N. V. Bennet A.J. PubMed Scopus Google Scholar), which that the of in the is on the state of the acid. is the of in the covalent with two in the the covalent the of the the into the and this to two of these studies have that an important role in Chem. Biol. 1996; 3: Full Text PDF PubMed Scopus Google Scholar, C. J. A. Boons G.J. J. Am. Chem. Soc. PubMed Scopus Google Scholar). The of five in the covalent be to the and the of the It has been suggested that that is at an can to to G.A. A. G. Taylor PubMed Scopus Google Scholar). This in to the covalent intermediate and may to the to product to with other sialidases, is as a on the but the of sialic acid. It may this of the active site from and to the of the by and to the is an inhibitor of sialidases, with All bacterial and sialidases have been in with this (7Crennell S.J. Garman E.F. Laver W.G. Vimr E.R. Taylor G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9852-9856Crossref PubMed Scopus (236) Google Scholar, 8Crennell S.J. Garman E. Laver G. Vimr E.R. Taylor G. Structure (Camb.). 1994; 2: 535-544Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 9Gaskell A. Crennell S.J. Taylor G. Structure (Camb.). 1995; 3: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 11Burmeister W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar, Y. Li Li Lou M. J. Mol. Biol. 1998; Scholar), as have the trypanosomal sialidases from T. (13Buschiazzo A. Tavares G.A. Campetella O. Spinelli S. Cremona M.L. Paris G. Amaya M.F. Frasch A.C. Alzari P.M. EMBO J. 2000; 19: 16-24Crossref PubMed Scopus (122) Google Scholar) and the from T. A. Amaya M.F. Cremona M.L. Frasch A.C. Alzari P.M. Mol. Cell. 2002; 10: 757-768Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar). These complexes have the active of these enzymes and in the of the by the transition-state It was that sialidases to form as a of their catalytic mechanism W.P. Henrissat B. Bosso C. Cusack C. Ruigrok R.W.H. Structure (Camb.). 1993; 1: 19-26Abstract Full Text PDF PubMed Scopus (152) Google Scholar, I. Connaris H. Taylor M. Zaitsev V. Wilson J.C. Kiefel M.J. von Itzstein M. Taylor G. J. Biol. Chem. 2004; 279: 40819-40826Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). This that the is that NanI is able to hydrate the of to form as a inhibitor of the influenza and as such a to be J.N. Colman P.M. 1992; PubMed Scopus Google Scholar). this is the of in the active site of a bacterial sialidase, which to have high in with the influenza virus are to be by retaining to a product a via a intermediate G. Chem. Biochem. PubMed Scopus Google Scholar). studies in that from the same as the catalytic acid R. PubMed Scopus Google Scholar, H. PubMed Scopus Google Scholar). it that addition of the acid the in a The to has been the of by a retaining 1994; PubMed Scopus Google Scholar). that on the and was by the The was a of the mechanism for such in which the as an and the with of an ion This via at the anomeric It was of to the of by the NanI sialidases it also not a acid but is not which The show that is to by NanI in the show that in this from the in this the its is as was the with the it is that the acid this This the that the provide acid catalysis and in the that a acid of the of for NanI a of and at and not The of in the crystal at be to a for at The mechanism for the of proceed as shown in The of the with the may to in the the in the the positioned on in an addition the of with the from the anomeric to of is This in a with the positive charge between the anomeric and the The structure of the covalent with NanI that such an intermediate be a covalent with the an to the positive charge on the anomeric a intermediate of a from this to the product These studies have a into the catalytic of a sialidase at an of that to of this that a key role in the of important the at the and the for to the The of the also used to complete this for

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.