Structural and Functional Characterization of PseC, an Aminotransferase Involved in the Biosynthesis of Pseudaminic Acid, an Essential Flagellar Modification in Helicobacter pylori

Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-α-d-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-β-l-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-β-l-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the β-l-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway. Helicobacter pylori flagellin is heavily glycosylated with the novel sialic acid-like nonulosonate, pseudaminic acid (Pse). The glycosylation process is essential for assembly of functional flagellar filaments and consequent bacterial motility. Because motility is a key virulence factor for this and other important pathogens, the Pse biosynthetic pathway offers potential for novel therapeutic targets. From recent NMR analyses, we determined that the conversion of UDP-α-d-Glc-NAc to the central intermediate in the pathway, UDP-4-amino-4,6-dideoxy-β-l-AltNAc, proceeds by formation of UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose by the dehydratase/epimerase PseB (HP0840) followed with amino transfer by the aminotransferase, PseC (HP0366). The central role of PseC in the H. pylori Pse biosynthetic pathway prompted us to determine crystal structures of the native protein, its complexes with pyridoxal phosphate alone and in combination with the UDP-4-amino-4,6-dideoxy-β-l-AltNAc product, the latter being converted to the external aldimine form in the active site of the enzyme. In the binding site, the AltNAc sugar ring adopts a 4C1 chair conformation, which is different from the predominant 1C4 form found in solution. The enzyme forms a homodimer where each monomer contributes to the active site, and these structures have permitted the identification of key residues involved in stabilization, and possibly catalysis, of the β-l-arabino intermediate during the amino transfer reaction. The essential role of Lys183 in the catalytic event was confirmed by site-directed mutagenesis. This work presents for the first time a nucleotide-sugar aminotransferase co-crystallized with its natural ligand, and, in conjunction with the recent functional characterization of this enzyme, these results will assist in elucidating the aminotransferase reaction mechanism within the Pse biosynthetic pathway. Flagellar glycosylation is common among a number of bacterial pathogens and has been implicated in immune avoidance, host/pathogen interactions, as well as in flagellar function and assembly (1Arora S.K. Neely A.N. Blair B. Lory S. Ramphal R. Infect. Immun. 2005; 73: 4395-4398Crossref PubMed Scopus (140) Google Scholar, 2Logan S.M. Kelly J.F. Thibault P. Ewing C.P. Guerry P. Mol. Microbiol. 2002; 46: 587-597Crossref PubMed Scopus (121) Google Scholar, 3Goon S. Kelly J.F. Logan S.M. Ewing C.P. Guerry P. Mol. Microbiol. 2003; 50: 659-671Crossref PubMed Scopus (152) Google Scholar, 4Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (224) Google Scholar). In Helicobacter pylori and Campylobacter jejuni, glycosylation of flagellin is required for the assembly of functional flagella, making its biosynthetic pathway a possible target for therapeutic intervention. Of particular interest are enzymes playing key roles in these pathways. Recently, mass spectrometry analysis of the H. pylori flagellar filament demonstrated that each flagellin monomer is modified with a novel nine-carbon sugar, pseudaminic acid (Pse) 2The abbreviations used are: Pse, 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (also known as pseudaminic acid); AAT, aspartate aminotransferase type I family; AltNAc, N-acetyl-β-L-altrosamine; PLP, pyridoxal phosphate; PMP, pyridoxamine phosphate; r.m.s.d., root mean square deviation. , or 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (4Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (224) Google Scholar). Single Pse moieties are attached via O-linkages to both the FlaA and FlaB monomeric structural proteins, at up to 7 and 10 Ser/Thr residues, respectively. The flagella of C. jejuni are similarly decorated but with more complex pseudaminic acids, including ones with acetamidino groups (2Logan S.M. Kelly J.F. Thibault P. Ewing C.P. Guerry P. Mol. Microbiol. 2002; 46: 587-597Crossref PubMed Scopus (121) Google Scholar, 4Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (224) Google Scholar, 5Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). This makes the H. pylori system more straightforward for analysis of the Pse pathway, although the biosynthetic genes are distributed around the genome, unlike the clustering within the flagellar glycosylation locus seen in C. jejuni (6Szymanski C.M. Logan S.M. Linton D. Wren B.W. Trends Microbiol. 2003; 11: 233-238Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The biosynthetic pathway for Pse has been established in outline, based on 1) characterization of UDP-linked intermediates that accumulate in enzyme knockouts in C. jejuni (7Soo E.C. Aubry A.J. Logan S.M. Guerry P. Kelly J.F. Young N.M. Thibault P. Anal. Chem. 2004; 76: 619-626Crossref PubMed Scopus (58) Google Scholar), 2) recent characterization of UDP-α-d-GlcNAc-modifying dehydratase/aminotransferase pairs from H. pylori and C. jejuni (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar), 3) the presence of homologs of the biosynthetic genes for the related nine-carbon sugar, sialic acid, in the flagellar locus of C. jejuni (9Guerry P. Doig P. Alm R.A. Burr D.H. Kinsella N. Trust T.J. Mol. Microbiol. 1996; 19: 369-378Crossref PubMed Scopus (91) Google Scholar, 10Parkhill J. Wren B.W. Mungall K. Ketley J.M. Churcher C. Basham D. Chillingworth T. Davies R.M. Feltwell T. Holroyd S. Jagels K. Karlyshev A.V. Moule S. Pallen M.J. Penn C.W. Quail M.A. Rajandream M.A. Rutherford K.M. van Vliet A.H. Whitehead S. Barrell B.G. Nature. 2000; 403: 665-668Crossref PubMed Scopus (1570) Google Scholar) and within the genome of H. pylori (11Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3027) Google Scholar, 12Alm R.A. Ling L.S. Moir D.T. King B.L. Brown E.D. Doig P.C. Smith D.R. Noonan B. Guild B.C. deJonge B.L. Carmel G. A. M. C. R. D. Trust T.J. Nature. PubMed Scopus Google Scholar), and the of Pse as determined by structural analysis P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). The biosynthetic pathway for sialic acid is by the and of to by the enzyme. This is followed by of this with by to which is with by In a the biosynthetic pathway of Pse by the of from by enzymes to and the of is common in the of complex bacterial in and the conversion is by a dehydratase/aminotransferase enzyme A. A. PubMed Scopus Google Scholar). The dehydratase/epimerase PseB (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) the first intermediate in the Pse biosynthetic pathway, UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose from enzyme with aminotransferase was to the reaction the transfer of amino in a pyridoxal phosphate to form UDP-4-amino-4,6-dideoxy-β-l-AltNAc (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) The PseC of enzymes to which a of involved in from of to of amino and P. of Mol. Biol. Scholar, Biol. 1996; Scopus Google Scholar). structures of nucleotide-sugar have been B.W. J.M. J. J. J. M.D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) and as of these structures was determined as complexes with sugar or found in the modified of of J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). has been as the enzyme for during the of in C. jejuni (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). we the crystal of PseC from H. pylori and its complexes with alone as well as the external aldimine of and the reaction product, In we residues important in the of this external aldimine intermediate and determine the of the in the PseC active we the role of PseC in flagellar glycosylation and H. pylori and pylori (11Tomb J.F. White O. Kerlavage A.R. Clayton R.A. Sutton G.G. Fleischmann R.D. Ketchum K.A. Klenk H.P. Gill S. Dougherty B.A. Nelson K. Quackenbush J. Zhou L. Kirkness E.F. Peterson S. Loftus B. Richardson D. Dodson R. Khalak H.G. Glodek A. McKenney K. Fitzegerald L.M. Lee N. Adams M.D. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3027) Google Scholar) used for the was from R. A. H. pylori from P. and from K. Helicobacter at on in a for of was by from of The was and confirmed by of the was to the by Logan S.M. M.A. Wakarchuk W.W. E. Mol. Microbiol. 2000; PubMed Scopus Google Scholar). of H. pylori and by natural as by R. van Mol. Microbiol. PubMed Scopus Google Scholar). was used in and the of the of the was confirmed by Helicobacter for motility by of with and or the 10 with or of the PseC and was the to the with (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) as the we used and for the we used and The as (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). H. pylori PseC functional and to (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) from the the (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) or the by and the was and at was by and and by a and dehydratase/epimerase from C. jejuni, was used to the PseC UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose as (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). analyses, of H. pylori was at in of PLP, 10 and of the analysis of PseC of PLP, 10 of H. pylori and of H. pylori or in at for of reaction by was similarly to that by (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). was determined the of and with the was to in with the of crystal of was in at from a by of in with of The to with a and and the complex with PLP, in and 10 was with to the of the complex at from a of and of by solution. of the complex to the with a and respectively. The complex was by of PseC in of in 10 The PseC was as (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and UDP-4-amino-4,6-dideoxy-β-l-AltNAc and in the of as well as of and of this complex by by of with of and the a solution. to the with a and and at the at a or a or with a on a The 1997; 276: PubMed Scopus Google Scholar). The was by with the D. Biol. 2001; PubMed Scopus Google Scholar) from the E. P. M. Dodson E. D. Biol. 2003; PubMed Scopus Google Scholar), the of as the of the was the A. Dodson D. Biol. PubMed Scopus Google Scholar), with used in the The of PseC with was the native as a The in the form of aldimine was in the and was in the the of PseC co-crystallized with and its product, UDP-4-amino-4,6-dideoxy-β-l-AltNAc was to with the native as the The for and for the but at for The that UDP-4-amino-4,6-dideoxy-β-l-AltNAc forms a with external The for the and of the ring was the for the of the and are in and have been with the G. H. J. Biol. 2000; 7 PubMed Scopus Google Scholar) with and related of the with the and with the T. C. J. Mol. Biol. PubMed Scopus Google Scholar). The of the sugar was determined by the A. Whitfield T. 2001; Scopus Google and of a of and of a in a of a that was involved in flagellar in H. pylori by The well and in or The motility of H. pylori in 1) and was determined by on the was as demonstrated by the of on motility In the the of of a H. pylori with the D. R. Mol. PubMed Scopus Google Scholar) the active motility of H. pylori PseC with the UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose of and are to that for the C. jejuni PseC (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The of the and at and on enzyme The H. pylori PseC and and in to the enzyme. and monomer of PseC as of The residues and its is a the with to the on by The and a of the of a from the and forms type with the other monomer of the The residues and is a with that of this and a that from the The the with the of its the of the of the and the the on the of the of of the monomer with and and and the involved in of the with the aldimine form of in the The at around is at the at around the to PseC at the PseC is a homodimer in by as well as in the crystal where the are related by of the of each monomer to of the of a and are at the and are the of the active are involved in the active of the which are by The are of from the of both up and from the other The the with the of that which the central with of the of the other The of and which with the other The at the are by 10 and of with the and external aldimine complexes that the binding of the in the protein, the being in the of in the of the where is in the native but the active site in the This to a a The of PseC in the presence of for the to the active site Lys183 in the form of aldimine to The site is at a the of the of the central and the of and The ring of is at the of this the phosphate is the residues from the of the to each The active site attached to PLP, is in the and This of the is that its the central The of the phosphate of with the of and and the of and of the first as well as with the of and, a with the of and from the This phosphate is the of to the on the phosphate The ring of is by on the and on the is within from the of the of Lys183 and the of The of is within from the of the of the The of is by of the with a in the aspartate aminotransferase type enzyme for the in a R.A. PubMed Scopus Google Scholar). is within and its is in a by to and of PseC with the reaction UDP-4-amino-4,6-dideoxy-β-l-AltNAc and to a different and crystal form with and the and and a of The of the complex to and UDP-4-amino-4,6-dideoxy-β-l-AltNAc in each of the active of the PseC and the presence of a the of the and of the that the external aldimine The of this intermediate that these the reaction in the to this The for the is that for a of this or the factor for within a of or the sugar is for the is for in the is for the sugar is In the for the and of the the of in the possible for this that the ring is and is known in to although the of the in the active site is in a 4C1 In the sugar ring is in with a of and ring (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. Chem. 2006; 281: 723-732Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). The within the to the and is by with residues from both of the on residues and and the on the of the the and and to the of the The ring residues from the the ring of and on the other is by and The is in a the and makes van with the are in this The is to the of of PLP, and a to the of and The presence of on the AltNAc ring by residues of the ring that are essential for aminotransferase Of possible for is the presence of well in the of the of is to the phosphate and the of and and is from the the other is to the ring of the AltNAc, to a and to the of The form to the of and and of the makes from to The with the in a at the and forms to the is to the groups of and and the is to the of The makes to the and van with and the glycosylation of flagellin from a number of important bacterial pathogens has (1Arora S.K. Neely A.N. Blair B. Lory S. Ramphal R. Infect. Immun. 2005; 73: 4395-4398Crossref PubMed Scopus (140) Google Scholar, 4Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (224) Google Scholar, 5Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, M. S.K. A. Vinogradov E. Thibault P. Ramphal R. Logan S.M. J. 2004; PubMed Scopus Google Scholar, M. M. Aubry A. Thibault P. M. Logan S.M. J. 2004; PubMed Scopus Google Scholar, S.K. M. Lory S. Ramphal R. S. A. 2001; PubMed Scopus Google Scholar), the functional and structural characterization of the involved in this process is in its we the crystal of a key enzyme involved in the of pseudaminic In the external aldimine complex the first of a nucleotide-sugar aminotransferase with its natural In we have the functional role of PseC in flagellar motility. The of PseC the within the of in the T. C. J. Mol. Biol. PubMed Scopus Google Scholar). PseC is in its to the aminotransferase from for followed by a enzyme from C. jejuni for and a enzyme, a from for Lee B.G. Chem. Biol. 2004; 11: Full Text Full Text PDF PubMed Scopus Google Scholar) have the sugar or with and on the structural and of and the of homologs in to PseC and are in and the amino in the 4C1 ring conformation, a and the amino in the 4C1 ring PseC and have different of homologs in with more homologs in in pathways. the structures for of of the sugar binding of the enzymes are in the of the binding and the residues that form residues, are among and including and and and and and In a characterization of binding site residues, was by site-directed to the found in at this in of the homologs of PseC in other but is in of the homologs of and are the common residues in these The its and the product, this is a of PseC a sugar being in the PseC is possible to the of the amino is and the are by PseC and the structural and of PseC and and to the of The is a which the of The of was determined in complex with which forms external aldimine with B.W. J.M. J. J. J. 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The of the structures that the The of the binding site is in both these and the of the in with the making to the and first the AltNAc sugar the the site in that the of the in around the sugar is in PseC and that a from a to to the of the AltNAc, which is in in The of this related to a in and of the to the This in a of of to residues in PseC the as well as a different of the latter is in from the monomer of the binding these enzymes are of of by in amino acid that are from the active The site residues of the The active site in and the aspartate to the of in are The the phosphate with in is and residues the active site in the The residues the active site are within the for the and residues and in The residues the at the active site are in in and in acid but is in the in In of and that of the binding site and in are and a role in binding and both are well in the different in the of and the is in The PseC enzyme the transfer of amino at of UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose the 4C1 which is a key in Pse This enzyme a mechanism with as the amino The reaction is in to that for B.W. J.M. J. J. J. M.D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). are the first is a type aminotransferase which results in The proceeds the formation of external aldimine intermediate with the sugar in a 4C1 ring conformation, for which we have determined the This type of intermediate was for B.W. J.M. J. J. J. M.D. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) but has been R.A. 2003; PubMed Scopus Google Scholar). The of this external aldimine intermediate a reaction with Lys183 to the The of as aminotransferase results have been for its in C. jejuni C. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). The recent characterization of the of PseB and PseC (8Schoenhofen I.C. McNally D.J. Vinogradov E. Whitfield D. Young M. Dick S. Wakarchuk W.W. Brisson J.-R. Logan S.M. J. Biol. 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