Artigo Acesso aberto Revisado por pares

Functional Characterization of Dehydratase/Aminotransferase Pairs from Helicobacter and Campylobacter

2005; Elsevier BV; Volume: 281; Issue: 2 Linguagem: Inglês

10.1074/jbc.m511021200

ISSN

1083-351X

Autores

Ian C. Schoenhofen, David J. McNally, Evgeny Vinogradov, Dennis M. Whitfield, N. Martin Young, Scott Dick, Warren W. Wakarchuk, Jean‐Robert Brisson, Susan M. Logan,

Tópico(s)

Enzyme Production and Characterization

Resumo

Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-d-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His6-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-l-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-d-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-d-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose. Helicobacter pylori and Campylobacter jejuni have been shown to modify their flagellins with pseudaminic acid (Pse), via O-linkage, while C. jejuni also possesses a general protein glycosylation pathway (Pgl) responsible for the N-linked modification of at least 30 proteins with a heptasaccharide containing 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose, a derivative of bacillosamine. To further define the Pse and bacillosamine biosynthetic pathways, we have undertaken functional characterization of UDP-α-d-GlcNAc modifying dehydratase/aminotransferase pairs, in particular the H. pylori and C. jejuni flagellar pairs HP0840/HP0366 and Cj1293/Cj1294, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c using His6-tagged purified derivatives. The metabolites produced by these enzymes were identified using NMR spectroscopy at 500 and/or 600 MHz with a cryogenically cooled probe for optimal sensitivity. The metabolites of Cj1293 (PseB) and HP0840 (FlaA1) were found to be labile and could only be characterized by NMR analysis directly in aqueous reaction buffer. The Cj1293 and HP0840 enzymes exhibited C6 dehydratase as well as a newly identified C5 epimerase activity that resulted in the production of both UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. In contrast, the Pgl dehydratase Cj1120c (PglF) was found to possess only C6 dehydratase activity generating UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. Substrate-specificity studies demonstrated that the flagellar aminotransferases HP0366 and Cj1294 utilize only UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose as substrate producing UDP-4-amino-4,6-dideoxy-β-l-AltNAc, a precursor in the Pse biosynthetic pathway. In contrast, the Pgl aminotransferase Cj1121c (PglE) utilizes only UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose producing UDP-4-amino-4,6-dideoxy-α-d-GlcNAc (UDP-2-acetamido-4-amino-2,4,6-trideoxy-α-d-glucopyranose), a precursor used in the production of the Pgl glycan component 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose. Campylobacter jejuni, a principle cause of acute gastroenteritis, and Helicobacter pylori, a major etiological agent of gastroduodenal disease, are microaerophilic Gram-negative bacteria (1Butzler J.P. Skirrow M.B. Clin. Gastroenterol. 1979; 8: 737-765Crossref PubMed Google Scholar, 2Dunn B.E. Cohen H. Blaser M.J. Clin. Microbiol. Rev. 1997; 10: 720-741Crossref PubMed Google Scholar). As these organisms have significant medical and public health importance, recent genomic efforts have resulted in the complete sequencing of at least two genomes for each organism with others in progress (3Parkhill 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 (1583) Google Scholar, 4Fouts D.E. Mongodin E.F. Mandrell R.E. Miller W.G. Rasko D.A. Ravel J. Brinkac L.M. Deboy R.T. Parker C.T. Daugherty S.C. Dodson R.J. Durkin A.S. Madupu R. Sullivan S.A. Shetty J.U. Ayodeji M.A. Shvartsbeyn A. Schatz M.C. Badger J.H. Fraser C.M. Nelson K.E. PloS. Biol. 2005; 3: e15Crossref PubMed Scopus (432) Google Scholar, 5Tomb 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. Hickey E.K. Berg D.E. Gocayne J.D. Utterback T.R. Peterson J.D. Kelley J.M. Cotton M.D. Weidman J.M. Fujii C. Bowman C. Watthey L. Wallin E. Hayes W.S. Borodovsky M. Karp P.D. Smith H.O. Fraser C.M. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3044) Google Scholar, 6Alm 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. Tummino P.J. Caruso A. Uria-Nickelsen M. Mills D.M. Ives C. Gibson R. Merberg D. Mills S.D. Jiang Q. Taylor D.E. Vovis G.F. Trust T.J. Nature. 1999; 397: 176-180Crossref PubMed Scopus (1607) Google Scholar). The sequencing of C. jejuni genomes revealed an unusual plethora (>8%) of glycan biosynthetic genes dedicated to surface carbohydrate biosynthesis, as well as significant diversity of glycan biosynthetic gene content among individual strains (7Karlyshev A.V. Ketley J.M. Wren B.W. FEMS Microbiol. Rev. 2005; 29: 377-390PubMed Google Scholar). These gene products appear to be involved in the biosynthesis of the glycoconjugate structures lipooligosaccharide and capsular polysaccharide, as well as in the biosynthesis of the novel glycans utilized by the N- and O-linked protein glycosylation systems. Each respective glycoconjugate structure is represented by a distinct and dedicated genetic locus (lipooligosaccharide Cj1131-Cj1152, capsule Cj1448c-Cj1413c, N-linked glycan Cj1119-Cj1130, O-linked glycan Cj1293-Cj1342). Although much progress has been made toward biosynthetic and structural characterization of lipooligosaccharide, capsular polysaccharide, and N-linked systems, the biochemical characterization of the O-linked system is currently less well defined (7Karlyshev A.V. Ketley J.M. Wren B.W. FEMS Microbiol. Rev. 2005; 29: 377-390PubMed Google Scholar, 8Szymanski 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, 9Szymanski C.M. Wren B.W. Nat. Rev. Microbiol. 2005; 3: 225-237Crossref PubMed Scopus (340) Google Scholar). C. jejuni has been shown to modify its flagellin with the novel nine-carbon sialic acid-like sugar 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid or pseudaminic acid (Pse), 2The abbreviations used are: Pse5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (also known as pseudaminic acid)AltNAcN-acetyl-β-l-altrosamineBac2,4-diamino-2,4,6-trideoxy-α-d-glucopyranose (also known as bacillosamine)CEcapillary electrophoresisδchemical shiftHMBCheteronuclear multiple bond coherenceHSQCheteronuclear single quantum coherenceJproton-coupling constantNOEnuclear Overhauser effectNOESYnuclear Overhauser effect spectroscopyPglgeneral protein glycosylation pathwayPLPpyridoxal phosphateTOCSYtotal correlation spectroscopy. as well as related derivatives, via O-linkage at up to 19 sites/monomer (10Logan S.M. Kelly J.F. Thibault P. Ewing C.P. Guerry P. Mol. Microbiol. 2002; 46: 587-597Crossref PubMed Scopus (121) Google Scholar, 11Thibault 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 (297) Google Scholar). The modification of flagellin appears to be important for flagellar assembly, as mutations in putative O-linked glycosylation genes result in non-motile cells lacking flagella (11Thibault 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 (297) Google Scholar). C. jejuni also possesses a general protein glycosylation pathway (Pgl) that is responsible for the N-linked addition of a heptasaccharide containing N-acetylgalactosamine, glucose, and 2,4-diacetamido-2,4,6-trideoxy-α-d-glucopyranose (2,4-diacetamido-Bac) to at least 30 different proteins (12Young N.M. Brisson J.R. Kelly J. Watson D.C. Tessier L. Lanthier P.H. Jarrell H.C. Cadotte N. St. Michael F. Aberg E. Szymanski C.M. J. Biol. Chem. 2002; 277: 42530-42539Abstract Full Text Full Text PDF PubMed Scopus (363) Google Scholar, 13Wacker M. Linton D. Hitchen P.G. Nita-Lazar M. Haslam S.M. North S.J. Panico M. Morris H.R. Dell A. Wren B.W. Aebi M. Science. 2002; 298: 1790-1793Crossref PubMed Scopus (649) Google Scholar). Interruption of either the flagellin glycosylation or Pgl pathway results in loss of colonization and, hence, virulence (14Black R.E. Levine M.M. Clements M.L. Hughes T.P. Blaser M.J. J. Infect. Dis. 1988; 157: 472-479Crossref PubMed Scopus (854) Google Scholar, 15Szymanski C.M. Yao R. Ewing C.P. Trust T.J. Guerry P. Mol. Microbiol. 1999; 32: 1022-1030Crossref PubMed Scopus (329) Google Scholar); as such, the Pse and Bac biosynthetic pathways offer potential as novel therapeutic targets. Although biosynthesis of both the complex carbohydrates Bac and Pse from the initial N-acetyl-hexosamine building block, UDP-α-d-GlcNAc, would involve the actions of a sugar-nucleotide dehydratase and its corresponding aminotransferase, the precise biosynthetic steps that distinguish these pathways are currently unknown. 5,7-diacetamido-3,5,7,9-tetradeoxy-l-glycero-l-manno-nonulosonic acid (also known as pseudaminic acid) N-acetyl-β-l-altrosamine 2,4-diamino-2,4,6-trideoxy-α-d-glucopyranose (also known as bacillosamine) capillary electrophoresis chemical shift heteronuclear multiple bond coherence heteronuclear single quantum coherence proton-coupling constant nuclear Overhauser effect nuclear Overhauser effect spectroscopy general protein glycosylation pathway pyridoxal phosphate total correlation spectroscopy. The flagellins of H. pylori have also been shown to be modified with Pse, where glycosylation again appears to be required for assembly of a functional filament (16Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (227) Google Scholar). Homologs of Campylobacter flagellar glycosylation components are present in both the H. pylori 26695 and the J99 genomes (5Tomb 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. Hickey E.K. Berg D.E. Gocayne J.D. Utterback T.R. Peterson J.D. Kelley J.M. Cotton M.D. Weidman J.M. Fujii C. Bowman C. Watthey L. Wallin E. Hayes W.S. Borodovsky M. Karp P.D. Smith H.O. Fraser C.M. Venter J.C. Nature. 1997; 388: 539-547Crossref PubMed Scopus (3044) Google Scholar, 6Alm 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. Tummino P.J. Caruso A. Uria-Nickelsen M. Mills D.M. Ives C. Gibson R. Merberg D. Mills S.D. Jiang Q. Taylor D.E. Vovis G.F. Trust T.J. Nature. 1999; 397: 176-180Crossref PubMed Scopus (1607) Google Scholar). Metabolomic analysis of various O-linked glycan biosynthetic mutants revealed an accumulation of biosynthetic intermediates and loss of CMP-Pse, thus confirming a role for these gene products in the Pse biosynthetic pathway, although the precise enzymatic steps of this pathway remain ill defined (16Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (227) Google Scholar). To unequivocally define the Bac and Pse biosynthetic pathways, we have undertaken functional characterization of the C. jejuni and H. pylori flagellar pairs Cj1293/Cj1294 and HP0840/HP0366, as well as the C. jejuni Pgl pair Cj1120c/Cj1121c, using His6-tagged recombinant proteins. These results convincingly demonstrate that the Pse and Bac biosynthetic pathways are discrete, incorporating unique and distinct dehydratase/aminotransferase pairs, and explain the clear phenotypic differences observed between isogenic mutants from the two pathways. DNA Techniques and Plasmid Construction—Plasmid DNA minipreparations and agarose gel purification of DNA fragments were performed using Qiagen's QIAprep spin kit and QIAquick gel extraction kit, respectively. All other recombinant DNA methods and analyses were performed as described by Sambrook et al. (17Sambrook J. Fritch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, New York1989Google Scholar). Vector or recombinant plasmids were transformed by electroporation into electrocompetent Top10F′ or DH10B (Invitrogen) Escherichia coli cells for cloning purposes or BL21[DE3] (Novagen, Madison, WI) E. coli cells for protein production. The polymerase chain reaction (PCR) was used to amplify H. pylori 26695 DNA or C. jejuni 11168 DNA for subsequent cloning. A list of cloning vectors and recombinant plasmids is provided in Table 1, and pertinent oligos are provided in Table 2. PCR was performed in a 50-μl reaction containing 10 mm Tris, pH 8.3, 50 mm KCl, 1.5 mm MgCl2, dNTPs at a final concentration of 200 μm, 0.2 μm of each primer, and 2.5 units of Taq DNA polymerase (Roche Applied Science). Amplicons were ligated with pCR2.1 and the agarose-purified restriction fragments were then ligated with either pET30a or pFO4 (a pET15b (Novagen) derivative in which the EcoRI-HindIII sites have been removed and replaced by sequence encoding MGSSHHHHHH). pET30a and pFO4 recombinant plasmids were sequenced using both forward and reverse T7 primers, as well as NRC175 and NRC160, respectively. Plasmid pNRC8.1 encodes a C-terminal His6-tagged derivative of HP0840 or FlaA1; pNRC37.1 encodes an N-terminal His6-tagged derivative of HP0366; pNRC20.3 encodes a C-terminal His6-tagged derivative of Cj1293 or PseB; pNRC82.1 encodes an N-terminal His6-tagged derivative of Cj1294; pNRC40.1 encodes an N-terminal His6-tagged soluble derivative of Cj1120c or PglF (residues 130–590); and pNRC41.3 encodes an N-terminal His6-tagged derivative of Cj1121c or PglE.TABLE 1Plasmids used in this studyPlasmidDescriptionSource/ReferencepCR2.1Apr, Knr, oriColE1, lac promoter, used for TA cloningInvitrogenpET30aKnr, oriColE1, T7 promoter, used for C-terminal His6-tagged protein expressionNovagenpFO4pET15b derivative; Apr, oriColE1, T7 promoter, used for N-terminal His6-tagged protein expressionThis studypNRC8.1HP0840 Ndel-Xhol in pET30a, encodes for HP0840His6This studypNRC37.1HP0366 BamHI-EcoRI in pFO4, encodes for His6HP0366This studypNRC20.3Cj1293 Ndel-Xhol in pET30a, encodes for Cj1293His6This studypNRC82.1Cj1294 BamHI-EcoRI in pFO4, encodes for His6Cj1294This studypNRC40.1Truncated Cj1120C BamHI-EcoRI in pFO4, encodes for His6SFCj1120cThis studypNRC41.3Cj1121c BamHI-EcoRI in pFO4, encodes for His6Cj1121cThis study Open table in a new tab TABLE 2Oligonucleotides used in this studyOligonucleotideSequence (5′ → 3′)PurposeNRC21CATATGCCAAATCATCAAAACATGCTAGACCloning of pNRC8.1NRC22CTCGAGTAATAATTTCAACAAATCATCAGGCTCNRC60GGATCCATGAAAGAGTTTGCTTATAGCloning of pNRC37.1NRC61AAAGAATTCTCATTCTATTTTAAAACTCTCAAAAGNRC43CATATGTTTAACAAAAAAAATATCTTAATCACGGCloning of pNRC20.3NRC44CTCGAGAAAACCTTCAGTATGATTGATGATTTCNRC137GGATCCATGCTTACTTATTCTCATCAAAACATCCloning of pNRC82.1NRC138GAATTCTTATCCACAATATCCCTTTTTAACTTTTTCNRC81GGATCCATGCTTGTGGATTTTAAACCTTCCloning of pNRC40.1NRC74GAATTCTTATACACCTTCTTTATTGTGTTTAAATTCNRC75GGATCCATGAGATTTTTTCTTTCTCCTCCGCloning of pNRC41.3NRC76GAATTCTTAAGCCTTTATGCTCTTTAAGATCT7-FTAATACGACTCACTATAGGGSequencing of pET30a constructsT7-RGCTAGTTATTGCTCAGCGGNRC175TTAATACGACTCACTATAGGGGAATTGSequencing of pFO4 constructsNRC160GGTTATGCTAGTTATTGCTCAGCGG Open table in a new tab His6-tagged Protein Purification—For functional characterization and for the isolation of enzyme products, each expression strain was grown in 500 ml of 2x yeast tryptone (17Sambrook J. Fritch E.F. Maniatis T. Molecular Cloning: a Laboratory Manual. 2nd Ed. Cold Spring Harbor Press, Cold Spring Harbor, New York1989Google Scholar) with either kanamycin (50 μg ml–1) or ampicillin (50 μgml–1) for selection. The cultures were grown at 30 °C, induced at an OD600 of 0.6 with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside, and harvested 2.75 h later. Cell pellets were resuspended in lysis buffer (50 mm sodium phosphate, pH 7.7, 400 mm NaCl, 10 mm β-mercaptoethanol) containing 10 mm imidazole and complete protease inhibitor mixture, EDTA-free (Roche Applied Science). After addition of 10 μgml–1 of RNaseA and DNaseI (Roche Applied Science), the cells were disrupted by two passes through an emulsiflex C5 (20,000 psi). Lysates were centrifuged at 100,000 × g for 1 h at 4°C, and the supernatant fraction was applied to a 1-ml nickel-nitrilotriacetic acid (Qiagen) column equilibrated in 10 mm imidazole lysis buffer, using a flow rate of 0.5 ml min–1. After sample application, the column was washed with 20 column volumes of 10 mm imidazole Lysis buffer. To elute the protein of interest, a linear gradient from 10 to 100 mm imidazole, in lysis buffer, over 60 column volumes was applied to the column prior to a final pulse of 40 column volumes of 200 mm imidazole lysis buffer. Fractions containing the purified protein of interest, as determined by SDS-PAGE (12.5%) and Coomassie staining, were pooled and dialyzed against dialysis buffer (25 mm sodium phosphate, pH 7.7, 50 mm NaCl) overnight at 4 °C. Protein concentration was measured spectrophotometrically using A280 0.1% values (HP0840His6, 0.536; His6HP0366, 0.386; Cj1293His6, 0.669; His6Cj1294, 0.635; His6SFCj1120c, 0.410; His6Cj1121c, 0.902). Enzymatic Reactions—Purification and dialysis/assay buffers for flagellar glycosylation enzymes were pH 7.2, whereas those for Pgl enzymes were pH 7.7. HP0840His6, Cj1293His6, and His6SFCj1120c reactions were scaled up to a total reaction volume of 20 ml containing 6 mg of enzyme in the presence of 1 mm UDP-α-d-GlcNAc. Time course samples were also taken for these dehydratase reactions at the times indicated; the samples were then boiled for 3 min, centrifuged for 3 min, and diluted 1:4 in H2O prior to capillary electrophoresis (CE) analysis. For the HP0840His6/His6HP0366 and the Cj1293His6/His6Cj1294 coupled assays, the reactions were scaled up to a total volume of 30 ml containing 9 mg of each enzyme in the presence of 1 mm UDP-α-d-GlcNAc, 10 mm l-Glu, and 1 mm pyridoxal phosphate (PLP) (the latter two being cofactors necessary for the aminotransfer reaction). To prepare the His6Cj1121c product, 25 ml of a His6SFCj1120c reaction containing 7.5 mg of enzyme in the presence of 1 mm UDP-α-d-GlcNAc was allowed to proceed for 210 min. After passage through an Amicon Ultra-15 (10,000 molecular weight cut-off) filter membrane, 6.3 mg of His6Cj1121c was added to the filtrate, along with PLP and l-Glu, to a final concentration of 1 and 10 mm, respectively. After incubation at 37 °C for 210 min, all reaction mixtures were passed through filter membranes as described above. The filtrates were then either 1) lyophilized and desalted using a Bio-Gel P-2 (Bio-Rad) column in 50 mm ammonium bicarbonate, pH 7.8, as well as a Sephadex G-15 column in pyridine:acetic acid:H2O (1:2.5:250) prior to NMR, or 2) analyzed directly by NMR in aqueous reaction buffer. Sample composition (i.e. distribution of products II-V, see Fig. 4) was analyzed by CE prior to NMR analysis. Analysis of Enzymatic Reaction Products by NMR Spectroscopy—Purified reaction products were suspended in 200 μl of 99% deuterated water (Cambridge Isotopes Laboratories Inc.). For examination of metabolites immediately after an enzymatic reaction was completed, the sample (90% H2O/10% deuterated water) was prepared by adding 20 μl of 99% deuterated water to 180 μl of reaction mixture. All samples were placed into 3-mm NMR tubes. Standard homo- and heteronuclear-correlated two-dimensional 1H NMR, 31P NMR, 13C HSQC, 31P HSQC, HMBC, COSY, TOCSY, NOESY pulse sequences from Varian (Varian, Palo Alto, CA) were used for general assignments. Selective one-dimensional TOCSY, with a Z-filter, and NOESY experiments were used for complete residue assignment and measurement of proton-coupling constants, JH,H, and NOEs (18Brisson J.R. Sue S.C. Wu W.G. McManus G. Nghia P.T. Uhrin D. Jimenez-Barbero J. Peters T. NMR Spectroscopy of Glycoconjugates. Wiley-VCH, Weinhem, Germany2002: 59-93Crossref Google Scholar, 19Uhrin D. Brisson J.-R. Barotin J. N Portais J. C NMR in Microbiology: Theory and Applications. Horizon Scientific Press, Wymonden, UK2000: 165-190Google Scholar). The analysis of JH,H, and NOE values for various pyranose chair forms was based on the coordinates for ideal pyranose chair conformations (20Berces A. Whitfield D.M. Nukada T. Tetrahedron. 2001; 57: 477-491Crossref Scopus (104) Google Scholar). NMR experiments were performed with a Varian 600 MHz (1H) spectrometer equipped with a Varian 5-mm Z-gradient triple resonance (1H, 13C, 15N) cryogenically cooled probe (cold probe) and with a Varian Inova 500 MHz (1H) spectrometer with a Varian Z-gradient 3-mm triple resonance (1H, 13C, 31P) probe. Direct detected 31P NMR spectra were acquired using a Varian Mercury 200 MHz (1H) spectrometer with a Nalorac 5-mm four nuclei probe. NMR experiments were typically performed at 25 °C with suppression of the H2O or deuterated HOD resonance at 4.78 ppm. For proton and carbon experiments, the methyl resonance of acetone was used as an internal reference (δH 2.225 ppm and δC 31.07 ppm), and an external 85% phosphoric acid standard (δP 0 ppm) was used to reference 31P spectra. Analysis of Reaction Products by CE—CE analysis was performed using a P/ACE 5510 system (Beckman Instruments) with diode array detection. The running buffer was 25 mm sodium tetraborate, pH 9.4. The capillary was either bare silica 50 μm × 50 cm or 75 μm × 50 cm, with a detector at 50 cm. The capillary was conditioned before each run by washing with 0.2 m NaOH for 2 min, water for 2 min, and running buffer for 2 min. Samples were introduced by pressure injection for 6–10 s, and the separation was performed at 18 kV for 20 min. Peak integration was done using the Beckman P/ACE station software. Kinetic Measurements of His6Cj1294 and His6Cj1121c—The substrates for the aminotransferases, UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose or UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose, were prepared by incubating UDP-α-d-GlcNAc with Cj1293His6. This substrate preparation was used because of its versatility in studying both Pse and Bac aminotransferases. To determine the preferred substrate for His6Cj1294 and His6Cj1121c, ∼15 μg of each enzyme was incubated with this Cj1293His6 filtrate, containing 10 mm l-Glu and 1 mm PLP, and subjected to time course analysis (see above). For kinetic analyses, 5 μg of His6 Cj1294 in a total volume of 200 μl, or 0.15 μg of His6Cj1121c in a total volume of 400 μl, was incubated at 37 °C in dialysis buffer, pH 7.2, containing 1 mm PLP, 10 mm l-Glu, and various quantities of UDP-2-acetamido-2,6-dideoxy-β-l-arabino-4-hexulose and UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose. His6Cj1121c assays also contained 40 μg of acetylated bovine serum albumin as carrier protein. Substrate conversion was determined using the molar extinction coefficient of UDP (ϵ260 = 10,000). Kinetic constants were calculated using Eadie-Hofstee plots with the program GraphPad Prism 3. Protein Expression and Purification—In this study, C. jejuni protein-coding sequences were cloned from C. jejuni 11168 DNA, and H. pylori sequences were cloned from strain 26695. Sugar nucleotide-modifying dehydratases were sensitive to N-terminal modification, whereas derivatives containing His6 tags located at the C terminus exhibited greater stability and solubility compared with their N-terminal counterparts (data not shown). As such, Cj1293 and its functional homolog HP0840 from H. pylori were both constructed with C-terminal His6 tags. In contrast, the Cj1120c derivative was designed with an N-terminal His6 tag, although, as described below, this was not adjacent to the catalytic domain. This derivative is a soluble truncated version of the native Cj1120c membrane protein, encoding only residues 130–590. From sequence alignment with the short soluble family of dehydratases the Cj1120c polypeptide can be divided into three domains: an N-terminal membrane domain containing four putative transmembrane regions, residues 1–129; a linker region, residues 130–267; and a functional or catalytic domain, residues 268–590 (data not shown). To assess the function of Cj1120c under native state conditions, the linker region was retained, although a construct containing only residues 189–590 exhibited similar activity to that observed with the 130–590 construct (data not shown). In contrast to the dehydratases, the aminotransferases HP0366, Cj1294, and Cj1121c were more resistant to N-terminal modification, but soluble yields were lower compared with the dehydratases (data not shown). Overall, yields of ∼20 mg/liter starting culture were obtained for all of the proteins tested, which had near homogeneity purity (Fig. 1). Protein production levels, as well as the correlation between size estimates determined by SDS-PAGE and that predicted from sequence, confirmed the identity of each purified His6-tagged protein. CE Characterization of HP0840, Cj1293, and Cj1120c Dehydratase Products—Similar to previously published reports of HP0840 (FlaA1) and Cj1293 (derived from C. jejuni subspecies doylei ATCC 49349) (21Creuzenet C. Schur M.J. Li J. Wakarchuk W.W. Lam J.S. J. Biol. Chem. 2000; 275: 34873-34880Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 22Creuzenet C. FEBS Lett. 2004; 559: 136-140Crossref PubMed Scopus (32) Google Scholar), we found that 11168 Cj1293His6 generated two products in sequential manner by CE. After ∼30 min of incubation of Cj1293His6 with UDP-α-d-GlcNAc the predominant product was peak IV (Fig. 2A), after which a shift occurred so that at 210 min the prominent product was peak II. In contrast, over the same time course, the only UDP-α-d-GlcNAc reaction product observed with His6SFCj1120c was peak II, which gradually increased over time (Fig. 2B). This is similar to the results obtained with WbpM, also a member of the large membrane-bound family of dehydratases (23Creuzenet C. Lam J.S. Mol. Microbiol. 2001; 41: 1295-1310Crossref PubMed Scopus (50) Google Scholar). Interestingly, there was a greater amount of the breakdown indicator, UDP, present in Cj1293 and HP0840 reactions compared with that observed with the Cj1120c reactions, suggesting that compound IV is less stable than the compound II (data not shown). Moreover, as seen with 49349 Cj1293, the dehydratases studied here did not require the addition of exogenous cofactor NAD(P)+, suggesting that the cofactor was already tightly bound within the enzymes and was recycled throughout catalysis. NMR Identification of the Products of Cj1120c and Cj1293/HP0840— Based on the results of one- and two-dimensional homo- and heteronuclear NMR experiments, the purified product of Cj1120c (II) was unambiguously identified as UDP-2-acetamido-2,6-dideoxy-α-d-xylo-4-hexulose or UDP-xylo-sugar. The 13C and 1H chemical shifts, as well as the proton-coupling constants determined for product II (Table 3), were identical to those previously reported for UDP-xylo-sugar (24Kneidinger B. O'Riordan K. Li J. Brisson J.R. Lee J.C. Lam J.S. J. Biol. Chem. 2003; 278: 3615-3627Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar) (supplemental Fig. S1).TABLE 3NMR chemical shifts δ (ppm) and coupling constants J (Hz) for metabolites of the bacillosamine and pseudaminic acid biosynthetic pathwaysCompound1H and 13C chemical shifts δ (ppm) and proton coupling constants J (Hz)H-1H-2H-3H-4H-5H-6NacC-1C-2C-3C-4C-5C-6CH3J(1,2)J(2,3)J(3,4)J(4,5)J(5,6)UDP-α-d-GlcNAc (I)δH5.513.993.813.553.923.82/3.862.08δC95.154.571.770.373.861.222.93JH,H3.310.210.210.13JH,P(β)7.3UDP-α-d-Xylo-sugar (II)δH5.454.103.824.121.242.07δC94.953.372.394.370.712.523.03JH,H3.410.86.53JH,P(β)7.0UDP-α-d-Bac (III)δH5.504.113.943.114.311.382.07δC95.054.667.958.066.617.622.83JH,H3.510.110.210.26.33JH,P(β)7.0UDP-β-l-Arabino-sugar (IV)δH5.554.243.924.061.372.07δC95.153.57

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