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Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168*

  • Myles B. Poulin
    Footnotes
    Affiliations
    Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Harald Nothaft
    Affiliations
    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Isabelle Hug
    Affiliations
    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Mario F. Feldman
    Footnotes
    Affiliations
    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Christine M. Szymanski
    Correspondence
    To whom correspondence may be addressed. Tel.: 780-248-1234; Fax: 780-492-9234;
    Affiliations
    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Todd L. Lowary
    Correspondence
    To whom correspondence may be addressed. Tel.: 780-492-1861; Fax: 780-492-7705;
    Affiliations
    Department of Chemistry, University of Alberta, Edmonton, Alberta T6G 2R3, Canada

    Alberta Ingenuity Centre for Carbohydrate Science, University of Alberta, Edmonton, Alberta T6G 2R3, Canada
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  • Author Footnotes
    * This work was supported in part by the Alberta Ingenuity Centre for Carbohydrate Science and National Sciences and Engineering Research Council of Canada.
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S2 and Tables S1–S2.
    1 Recipient of a studentship award from Alberta Ingenuity.
    2 Alberta Heritage Foundation for Medical Research Scholar.
Open AccessPublished:November 03, 2009DOI:https://doi.org/10.1074/jbc.M109.072157
      UDP-galactopyranose mutases (UGM) are the enzymes responsible for the synthesis of UDP-galactofuranose (UDP-Galf) from UDP-galactopyranose (UDP-Galp). The enzyme, encoded by the glf gene, is present in bacteria, parasites, and fungi that express Galf in their glycoconjugates. Recently, a UGM homologue encoded by the cj1439 gene has been identified in Campylobacter jejuni 11168, an organism possessing no Galf-containing glycoconjugates. However, the capsular polysaccharide from this strain contains a 2-acetamido-2-deoxy-d-galactofuranose (GalfNAc) moiety. Using an in vitro high performance liquid chromatography assay and complementation studies, we characterized the activity of this UGM homologue. The enzyme, which we have renamed UDP-N-acetylgalactopyranose mutase (UNGM), has relaxed specificity and can use either UDP-Gal or UDP-GalNAc as a substrate. Complementation studies of mutase knock-outs in C. jejuni 11168 and Escherichia coli W3110, the latter containing Galf residues in its lipopolysaccharide, demonstrated that the enzyme recognizes both UDP-Gal and UDP-GalNAc in vivo. A homology model of UNGM and site-directed mutagenesis led to the identification of two active site amino acid residues involved in the recognition of the UDP-GalNAc substrate. The specificity of UNGM was characterized using a two-substrate co-incubation assay, which demonstrated, surprisingly, that UDP-Gal is a better substrate than UDP-GalNAc.

      Introduction

      In nature, hexose sugars are found predominantly in the thermodynamically favored pyranose ring form; however, hexose sugars in the furanose ring form are found in bacteria, fungi, and parasites (
      • Peltier P.
      • Euzen R.
      • Daniellou R.
      • Nugier-Chauvin C.
      • Ferrières V.
      ,
      • Richards M.R.
      • Lowary T.L.
      ). For example, d-galactofuranose (Galf)
      The abbreviations used are: Galf
      d-galactofuranose
      Cj
      Campylobacter jejuni
      CPS
      capsular polysaccharide
      Ec
      Escherichia coli
      LPS
      lipopolysaccharide
      UGM
      UDP-galactopyranose mutase
      UNGM
      UDP-N-acetylgalactopyranose mutase
      Galp
      galactopyranose
      HPLC
      high pressure liquid chromatography.
      is a component in many microbial cell surface oligosaccharides (
      • de Lederkremer R.M.
      • Colli W.
      ,
      • Whitfield C.
      ) and is a major structural component of the mycobacterial cell wall (
      • Brennan P.J.
      • Nikaido H.
      ). In many pathogenic microorganisms, these Galf residues are essential for cell viability or play a crucial role in cell physiology (
      • Lee R.E.
      • Smith M.D.
      • Nash R.J.
      • Griffiths R.C.
      • McNeil M.
      • Grewal R.K.
      • Yan W.X.
      • Besra G.S.
      • Brennan P.J.
      • Fleet G.W.
      ,
      • Pan F.
      • Jackson M.
      • Ma Y.
      • McNeil M.
      ). For this reason, and because hexofuranose sugars are absent in mammalian cell saccharide structures (
      • Peltier P.
      • Euzen R.
      • Daniellou R.
      • Nugier-Chauvin C.
      • Ferrières V.
      ), there has been a surge of interest in studying and identifying inhibitors of Galf biosynthesis (
      • Pedersen L.L.
      • Turco S.J.
      ).
      The sugar nucleotide UDP-Galf is the precursor of Galf and is incorporated into growing oligosaccharides via galactofuranosyltransferase-mediated reactions (
      • Belánová M.
      • Dianisková P.
      • Brennan P.J.
      • Completo G.C.
      • Rose N.L.
      • Lowary T.L.
      • Mikusová K.
      ). First identified in Escherichia coli (
      • Nassau P.M.
      • Martin S.L.
      • Brown R.E.
      • Weston A.
      • Monsey D.
      • McNeil M.R.
      • Duncan K.
      ), the enzyme UDP-d-galactopyranose mutase (UGM) is responsible for the biosynthesis of UDP-Galf via the ring contraction of UDP-galactopyranose (UDP-Galp). UGM is encoded by the glf gene for which homologues have since been identified in Klebsiella pneumoniae (
      • Köplin R.
      • Brisson J.R.
      • Whitfield C.
      ), mycobacterial species (
      • Weston A.
      • Stern R.J.
      • Lee R.E.
      • Nassau P.M.
      • Monsey D.
      • Martin S.L.
      • Scherman M.S.
      • Besra G.S.
      • Duncan K.
      • McNeil M.R.
      ), and in various eukaryotic pathogens (
      • Beverley S.M.
      • Owens K.L.
      • Showalter M.
      • Griffith C.L.
      • Doering T.L.
      • Jones V.C.
      • McNeil M.R.
      ,
      • Bakker H.
      • Kleczka B.
      • Gerardy-Schahn R.
      • Routier F.H.
      ). Since the advent of rapid genome sequencing, a number of putative UGMs have been identified throughout the microbial species; however, very few of the gene products have been confirmed by biochemical analysis.
      UGM is a flavoprotein and catalyzes the reversible ring contraction of UDP-Galp to UDP-Galf via a unique mechanism (Fig. 1) (
      • Sanders D.A.
      • Staines A.G.
      • McMahon S.A.
      • McNeil M.R.
      • Whitfield C.
      • Naismith J.H.
      ). The noncovalently bound FAD co-factor is directly involved in catalysis and must be in the reduced form for the enzyme to be active (
      • Zhang Q.
      • Liu H.
      ). Because of the interest in UGM as a drug target (
      • Pedersen L.L.
      • Turco S.J.
      ), significant work has been done to study its mechanism, and it has been shown that the reduced FADH acts as a nucleophile and displaces the anomeric UDP to form a covalent intermediate (
      • Soltero-Higgin M.
      • Carlson E.E.
      • Gruber T.D.
      • Kiessling L.L.
      ). Formation of an iminium ion breaks the O5–C1 bond of the galactose moiety leading to a covalently bound acyclic intermediate. This species can then cyclize to the furanose ring form.
      Although the enzyme mechanism is generally understood, there are still many unanswered questions about the enzyme-substrate interactions. The UGM protein structure contains a mobile loop region, which adopts either an open or closed form in the crystal structures that have been determined to date (
      • Sanders D.A.
      • Staines A.G.
      • McMahon S.A.
      • McNeil M.R.
      • Whitfield C.
      • Naismith J.H.
      ,
      • Beis K.
      • Srikannathasan V.
      • Liu H.
      • Fullerton S.W.
      • Bamford V.A.
      • Sanders D.A.
      • Whitfield C.
      • McNeil M.R.
      • Naismith J.H.
      ) with the closed structure being the catalytically active form. This loop has been shown to close upon substrate binding (
      • Yao X.H.
      • Bleile D.W.
      • Yuan Y.
      • Chao J.
      • Sarathy K.P.
      • Sanders D.A.
      • Pinto B.M.
      • O'Neill M.A.
      ), and a conserved arginine (Arg-174 in K. pneumoniae, Arg-170 in E. coli, and Arg-180 in Mycobacterium tuberculosis) has been found to be essential for UGM activity (
      • Chad J.M.
      • Sarathy K.P.
      • Gruber T.D.
      • Addala E.
      • Kiessling L.L.
      • Sanders D.A.R.
      ). This arginine appears to stabilize the negatively charged diphosphate backbone of the sugar nucleotide substrate. Many synthetic analogues (
      • Caravano A.
      • Sinaÿ P.
      • Vincent S.P.
      ,
      • Itoh K.
      • Huang Z.
      • Liu H.W.
      ,
      • Carlson E.E.
      • May J.F.
      • Kiessling L.L.
      ,
      • Zhang Q.
      • Liu H.
      ,
      • Yuan Y.
      • Bleile D.W.
      • Wen X.
      • Sanders D.A.
      • Itoh K.
      • Liu H.W.
      • Pinto B.M.
      ,
      • Errey J.C.
      • Mann M.C.
      • Fairhurst S.A.
      • Hill L.
      • McNeil M.R.
      • Naismith J.H.
      • Percy J.M.
      • Whitfield C.
      • Field R.A.
      ) have been used to probe the mechanism of UGM and investigate substrate binding, but until recently, no ligand-bound crystal structures have been available. Tryptophan fluorescence (
      • Sanders D.A.
      • Staines A.G.
      • McMahon S.A.
      • McNeil M.R.
      • Whitfield C.
      • Naismith J.H.
      ) and molecular modeling have predicted that the uridine of the UDP-Gal base stacks with Trp-160 (numbering for K. pneumoniae) (
      • Yuan Y.
      • Wen X.
      • Sanders D.A.
      • Pinto B.M.
      ); in contrast, recent crystal structures of the K. pneumoniae UGM with bound UDP-Glcp (
      • Gruber T.D.
      • Borrok M.J.
      • Westler W.M.
      • Forest K.T.
      • Kiessling L.L.
      ) and UDP-Galp (
      • Gruber T.D.
      • Westler W.M.
      • Kiessling L.L.
      • Forest K.T.
      ) show that the uridine stacks against tyrosine 155 in the active site. This discrepancy demonstrates that many of the key binding interactions responsible for the substrate specificity of UGM still remain to be elucidated.
      Although Galf is the most common naturally occurring hexofuranose, it is not unique. 6-Deoxy-d-galactofuranose (
      • Feng L.
      • Senchenkova S.N.
      • Yang J.
      • Shashkov A.S.
      • Tao J.
      • Guo H.
      • Cheng J.
      • Ren Y.
      • Knirel Y.A.
      • Reeves P.R.
      • Wang L.
      ), 6-deoxy-l-altrofuranose (
      • Hanniffy O.M.
      • Shashkov A.S.
      • Moran A.P.
      • Prendergast M.M.
      • Senchenkova S.N.
      • Knirel Y.A.
      • Savage A.V.
      ), and 2-acetamido-2-deoxy-d-galactofuranose (
      • Arbatsky N.P.
      • Shashkov A.S.
      • Mamyan S.S.
      • Knirel Y.A.
      • Zych K.
      • Sidorczyk Z.
      ,
      • St Michael F.
      • Szymanski C.M.
      • Li J.
      • Chan K.H.
      • Khieu N.H.
      • Larocque S.
      • Wakarchuk W.W.
      • Brisson J.R.
      • Monteiro M.A.
      ), among others, have also been identified in bacterial saccharide structures. However, little is known about the biosynthesis of these other hexofuranose sugars. Previous work has established that the UGM from K. pneumoniae, which has been the most studied, is unable to catalyze the synthesis of either UDP-Fucf or UDP-GalfNAc (
      • Errey J.C.
      • Mann M.C.
      • Fairhurst S.A.
      • Hill L.
      • McNeil M.R.
      • Naismith J.H.
      • Percy J.M.
      • Whitfield C.
      • Field R.A.
      ,
      • Eppe G.
      • Peltier P.
      • Daniellou R.
      • Nugier-Chauvin C.
      • Ferrières V.
      • Vincent S.P.
      ). Recently it has been shown that a homologue of the glf gene, fcf2 in E. coli O52, acts as a Fucf mutase enzyme for the biosynthesis of TDP-Fucf (
      • Wang Q.
      • Ding P.
      • Perepelov A.V.
      • Xu Y.
      • Wang Y.
      • Knirel Y.A.
      • Wang L.
      • Feng L.
      ). This protein has 60% identity to the K. pneumoniae UGM, but the origin of the difference in substrate tolerance is unknown.
      The bacterium Campylobacter jejuni is a foodborne pathogen that is a leading cause of diarrheal disease worldwide (
      • Allos B.M.
      ). Infections by this organism have also been linked to the development of the neurological disorder Guillain-Barré syndrome (
      • Kaldor J.
      • Speed B.R.
      ). Previous work showed that the capsular polysaccharide (CPS) from the 11168 strain contains a GalfNAc residue (Fig. 2) (
      • St Michael F.
      • Szymanski C.M.
      • Li J.
      • Chan K.H.
      • Khieu N.H.
      • Larocque S.
      • Wakarchuk W.W.
      • Brisson J.R.
      • Monteiro M.A.
      ). C. jejuni 11168 also contains a homologue of the glf gene cj1439. Because no Galf residues have been found in C. jejuni 11168 glycoconjugates, it has been proposed that the cj1439 gene product is responsible for the biosynthesis of UDP-GalfNAc from UDP-GalpNAc (
      • St Michael F.
      • Szymanski C.M.
      • Li J.
      • Chan K.H.
      • Khieu N.H.
      • Larocque S.
      • Wakarchuk W.W.
      • Brisson J.R.
      • Monteiro M.A.
      ). Herein, we report studies on the protein produced by expression of cj1439 and demonstrate its activity as a UDP-N-acetylgalactopyranose mutase (UNGM). We also have demonstrated that the enzyme can use both UDP-Gal and UDP-GalNAc as substrates and have investigated the origins of this substrate selectivity using site-directed mutagenesis to identify key residues that allow for the turnover of UDP-GalNAc.
      Figure thumbnail gr2
      FIGURE 2Capsular polysaccharide structure of C. jejuni 11168. Shown is the tetrasaccharide repeat unit of C. jejuni 11168 (HS:2 serotype) with the GalfNAc residue shown in boldface.

      DISCUSSION

      In this paper, we have tested a previously proposed hypothesis (
      • Karlyshev A.V.
      • Champion O.L.
      • Churcher C.
      • Brisson J.R.
      • Jarrell H.C.
      • Gilbert M.
      • Brochu D.
      • St Michael F.
      • Li J.
      • Wakarchuk W.W.
      • Goodhead I.
      • Sanders M.
      • Stevens K.
      • White B.
      • Parkhill J.
      • Wren B.W.
      • Szymanski C.M.
      ) that the C. jejuni 11168 gene cj1439 encodes a protein responsible for the biosynthesis of the UDP-GalfNAc. Our investigations have shown that, unlike the highly homologous UGM from E. coli and K. pneumoniae, the C. jejuni enzyme is able to convert the sugar nucleotide UDP-GalpNAc to UDP-GalfNAc, the precursor of GalfNAc in the CPS. Thus, the protein is a UNGM. In addition, we have demonstrated that the C. jejuni UNGM also converts UDP-Galp to UDP-Galf both in vitro and in vivo in E. coli. We also identified two amino acids in the active site of UNGM that play a role in the interconversion of UDP-GalfNAc and UDP-GalpNAc. Our co-incubation assay allowed us to examine, in a single reaction, the substrate specificity of wild-type UNGM protein and each of the UNGM mutants.

      Cj1439 Encodes for a Protein with Both UGM and UNGM Activity

      This study represents the first demonstration of an enzyme involved in the interconversion of UDP-GalpNAc and UDP-GalfNAc. This enzyme, which bears a high sequence similarity to known UGMs, produces UDP-GalfNAc from UDP-GalpNAc presumably via a similar ring contraction mechanism. Both in vitro investigations and an in vivo complementation experiment support the role of UNGM in the biosynthesis of the GalfNAc in C. jejuni 11168. In addition, we have demonstrated that the C. jejuni UNGM has dual substrate specificity and can interconvert the furanose and pyranose isomers of both UDP-Gal and UDP-GalNAc. Furthermore, the UGM activity of the C. jejuni enzyme has been demonstrated in vivo where it is able to complement the activity of the E. coli UGM in a glf gene knock-out.
      It is not unusual that bacteria with compact genomes express enzymes that exhibit more than one activity. These bifunctional enzymes are widespread among bacteria and allow for the synthesis of many complex structures advantageous to the survival of the organism while still maintaining a small genome size. An example is the wbbO gene product from K. pneumoniae, a galactosyltransferase that catalyzes the transfer of both Galp and Galf residues in the biosynthesis of the lipopolysaccharide O1 antigen (
      • Guan S.
      • Clarke A.J.
      • Whitfield C.
      ). Bifunctional enzymes have also been characterized in C. jejuni. For example, a single UDP-GlcNAc/Glc 4-epimerase was shown to be involved in the biosynthesis of three cell surface glycoconjugates in strain 11168 (
      • Bernatchez S.
      • Szymanski C.M.
      • Ishiyama N.
      • Li J.
      • Jarrell H.C.
      • Lau P.C.
      • Berghuis A.M.
      • Young N.M.
      • Wakarchuk W.W.
      ). Because the CPS structures in C. jejuni are highly variable between serotypes, it is reasonable to hypothesize that a bifunctional UNGM would be advantageous. However, no Galf residues have been identified in any glycoconjugate from this organism. Thus, the inclusion of GalfNAc, instead of Galf, into the CPS appears to be due to the specificity of the cognate glycosyltransferase.

      Comparing Calculated Specificity of C. jejuni UNGM to the Experimentally Determined Specificity

      In this study, two methods were used to determine the substrate specificity of the C. jejuni UNGM. The first was the direct determination from the ratio of product conversion observed in the co-incubation assay (Fig. 7). The second was calculated using the observed kinetic parameters for each protein with UDP-Galf or UDP-GalfNAc (Table 1). The observed trend of substrate specificity is the same by either method; however, the calculated specificity in each case is lower than that determined using the co-incubation assay. The calculated substrate specificity may not accurately represent the observed specificity as the combined rate of the two competing reactions may be greater than, equal to, or lower than the rate of each individual reaction (
      • Cha S.
      ). Because of this, it appears that the co-incubation assay more accurately represents the substrate specificity of the UNGM for its two competing substrates UDP-Galf and UDP-GalfNAc.

      Effect of Two Active Site Arginines on UDP-GalNAc Recognition by UNGM

      We believed that residues in, or in proximity to, the active site would play a role in the different substrate specificity of the C. jejuni UNGM compared with other known UGM enzymes. We also rationalized that these key residues would be conserved in the other UGM but not in the C. jejuni enzyme. The only two residues that fit these criteria were Arg-59 and Arg-168. In both cases, these residues were found to be other basic amino acids, histidine and lysine, respectively, in the E. coli and K. pneumoniae UGM. In M. tuberculosis, Arg-59 was also replaced by histidine; however, Arg-168 was found to be a threonine rather than the lysine observed in E. coli and K. pneumoniae.
      Mutagenesis of R168K only resulted in a 2-fold increase in selectivity for UDP-Galf over UDP-GalfNAc in the co-incubation assay; however, there were more significant changes observed for the kinetic parameters. This amino acid change resulted in a decrease in the catalytic activity of the protein for both substrates compared with the wild-type UNGM, but most interesting was that this substitution resulted in a significant increase in the Km value with UDP-GalfNAc but not with UDP-Galf. As the Km value approximates the dissociation constant for the enzyme-substrate complex, this supports the notion that Arg-168 has a role in binding and stabilizing UDP-GalNAc in the active site. It has previously been proposed that amino acid residues in the mobile loop of UGMs are involved in substrate recognition (
      • Yao X.H.
      • Bleile D.W.
      • Yuan Y.
      • Chao J.
      • Sarathy K.P.
      • Sanders D.A.
      • Pinto B.M.
      • O'Neill M.A.
      ,
      • Chad J.M.
      • Sarathy K.P.
      • Gruber T.D.
      • Addala E.
      • Kiessling L.L.
      • Sanders D.A.R.
      ). As Arg-168 is located in the mobile loop, its ability to stabilize the UDP-GalNAc enzyme-substrate complex is consistent with the role of the mobile loop in substrate recognition. This arginine residue is located 4.2 Åfrom the acetamido carbonyl oxygen in our homology model with docked UDP-GalpNAc (Fig. 6A), indicating a possible hydrogen bonding interaction.
      Mutation of the other active site arginine (Arg-59) indicates that it also plays an important role in the catalytic activity of UNGM. Mutagenesis of R59H results in a greater than 4-fold decrease in kcat for UDP-GalfNAc, while simultaneously resulting in a 3-fold increase in kcat for UDP-Galf. The Km value is also changed for both substrates, but this appears minor in comparison with the observed changes in kcat. Considering our homology model, Arg-59 is located within 4.5 Åof the carbonyl oxygen of the acetamido group of UDP-GalpNAc (Fig. 6A). In the E. coli UGM crystal structure, the equivalent residue His-59 sits below the active site (data not shown) and does not protrude into the active site as does Arg-59 in UNGM. Therefore, it appears that Arg-59 is able to interact with the UDP-GalNAc substrate to stabilize the intermediate. This may occur by preventing the formation of nonproductive oxazoline-like intermediates, which could be formed by an intramolecular reaction of the acetamido group (Fig. 8). When the substrate is UDP-Gal, then there is no possibility of forming such intermediates. Therefore, Arg-59 does not aid in catalysis. Instead, because arginine is more bulky than histidine, it could lower the catalytic rate due to steric interactions in the active site during catalysis.
      Figure thumbnail gr8
      FIGURE 8Formation of oxazoline intermediate. A, acetamido group of GalNAc can undergo an unproductive intramolecular reaction to form an FAD-bound oxazoline intermediate that prevents conversion to GalfNAc. B, possible interaction between Arg-59 and the GalNAc acetamido group that would prevent formation of an oxazoline intermediate and promote conversion to GalfNAc.
      The mutagenesis of both R59H and R168K results in a larger decrease in turnover of UDP-GalfNAc substrate while simultaneously increasing turnover of UDP-Galf. This results in an increased selectivity for UDP-Galf than observed for either of the single mutants (Fig. 7; Table 1). Somewhat surprisingly, the increase in the Km value for UDP-GalfNAc seen for the R168K mutant was not observed in the case of the double mutant. This suggests that the amino acids play a synergistic role in allowing the enzyme to interconvert UDP-GalfNAc and UDP-GalpNAc, rather than an additive role in which Arg-59 is the major determinant.

      Subtle Amino Acid Substitutions Result in Changes in C. jejuni UNGM Substrate Tolerance

      Despite the high sequence identity of C. jejuni UNGM with the E. coli, M. tuberculosis, and K. pneumoniae UGM, the UNGM possesses the ability to recognize UDP-GalNAc as a substrate. Most of the sequence variability occurs in solvent-exposed residues, and the amino acids making up the active site are nearly identical in all four mutase enzymes (supplemental Fig. S2, boxed residues). The two residues identified here as playing an important role in allowing recognition of UDP-GalNAc are relatively conservative replacements of the residues found in the other mutase enzymes; however, they nevertheless have a significant effect on the substrate selectivity of the enzyme. It should be appreciated that small variations in amino acids leading to changes in substrate specificity are well known in carbohydrate-active enzymes. For example, the blood group GTA and GTB glycosyltransferases, which use UDP-GalpNAc versus UDP-Galp, respectively, as the donor substrate, differ in only four amino acids (
      • Rose N.L.
      • Palcic M.M.
      • Evans S.V.
      ). Similarly, in Neisseria meningitidis, a single amino acid change in the capsule polymerase determines the substrate specificity for either Glcp or Galp transferase activity (
      • Claus H.
      • Stummeyer K.
      • Batzilla J.
      • Mühlenhoff M.
      • Vogel U.
      ).
      Although the two arginine residues identified in this study influence the substrate specificity, the mutagenesis of either residue or both failed to result in a complete loss in UNGM activity. It is therefore clear that other amino acids further removed from the active site also contribute to the specificity of the enzyme, and these remain to be elucidated. As well, we have demonstrated that C. jejuni UNGM can function as a UGM in vivo; however, there have been no Galf residues reported in C. jejuni glycoconjugates. In this context, another unresolved issue is if other C. jejuni strains containing the glf gene possess Galf-containing glycoconjugates, and investigations into the specificity of the transferases accepting donors from bifunctional enzymes are warranted. This study further demonstrates the intricacies in bacterial glycoconjugate biosynthesis. Detailed understanding of these systems will allow for the development of novel antimicrobials targeting these pathogen-specific pathways.

      REFERENCES

        • Peltier P.
        • Euzen R.
        • Daniellou R.
        • Nugier-Chauvin C.
        • Ferrières V.
        Carbohydr. Res. 2008; 343: 1897-1923
        • Richards M.R.
        • Lowary T.L.
        ChemBioChem. 2009; 10: 1920-1938
        • de Lederkremer R.M.
        • Colli W.
        Glycobiology. 1995; 5: 547-552
        • Whitfield C.
        Trends Microbiol. 1995; 3: 178-185
        • Brennan P.J.
        • Nikaido H.
        Annu. Rev. Biochem. 1995; 64: 29-63
        • Lee R.E.
        • Smith M.D.
        • Nash R.J.
        • Griffiths R.C.
        • McNeil M.
        • Grewal R.K.
        • Yan W.X.
        • Besra G.S.
        • Brennan P.J.
        • Fleet G.W.
        Tetrahedron Lett. 1997; 38: 6733-6736
        • Pan F.
        • Jackson M.
        • Ma Y.
        • McNeil M.
        J. Bacteriol. 2001; 183: 3991-3998
        • Pedersen L.L.
        • Turco S.J.
        Cell. Mol. Life Sci. 2003; 60: 259-266
        • Belánová M.
        • Dianisková P.
        • Brennan P.J.
        • Completo G.C.
        • Rose N.L.
        • Lowary T.L.
        • Mikusová K.
        J. Bacteriol. 2008; 190: 1141-1145
        • Nassau P.M.
        • Martin S.L.
        • Brown R.E.
        • Weston A.
        • Monsey D.
        • McNeil M.R.
        • Duncan K.
        J. Bacteriol. 1996; 178: 1047-1052
        • Köplin R.
        • Brisson J.R.
        • Whitfield C.
        J. Biol. Chem. 1997; 272: 4121-4128
        • Weston A.
        • Stern R.J.
        • Lee R.E.
        • Nassau P.M.
        • Monsey D.
        • Martin S.L.
        • Scherman M.S.
        • Besra G.S.
        • Duncan K.
        • McNeil M.R.
        Tuber Lung Dis. 1997; 78: 123-131
        • Beverley S.M.
        • Owens K.L.
        • Showalter M.
        • Griffith C.L.
        • Doering T.L.
        • Jones V.C.
        • McNeil M.R.
        Eukaryotic Cell. 2005; 4: 1147-1154
        • Bakker H.
        • Kleczka B.
        • Gerardy-Schahn R.
        • Routier F.H.
        Biol. Chem. 2005; 386: 657-661
        • Sanders D.A.
        • Staines A.G.
        • McMahon S.A.
        • McNeil M.R.
        • Whitfield C.
        • Naismith J.H.
        Nat. Struct. Biol. 2001; 8: 858-863
        • Zhang Q.
        • Liu H.
        J. Am. Chem. Soc. 2000; 122: 9065-9070
        • Soltero-Higgin M.
        • Carlson E.E.
        • Gruber T.D.
        • Kiessling L.L.
        Nat. Struct. Mol. Biol. 2004; 11: 539-543
        • Beis K.
        • Srikannathasan V.
        • Liu H.
        • Fullerton S.W.
        • Bamford V.A.
        • Sanders D.A.
        • Whitfield C.
        • McNeil M.R.
        • Naismith J.H.
        J. Mol. Biol. 2005; 348: 971-982
        • Yao X.H.
        • Bleile D.W.
        • Yuan Y.
        • Chao J.
        • Sarathy K.P.
        • Sanders D.A.
        • Pinto B.M.
        • O'Neill M.A.
        Proteins Struct. Funct. Bioinformat. 2009; 74: 972-979
        • Chad J.M.
        • Sarathy K.P.
        • Gruber T.D.
        • Addala E.
        • Kiessling L.L.
        • Sanders D.A.R.
        Biochemistry. 2007; 46: 6723-6732
        • Caravano A.
        • Sinaÿ P.
        • Vincent S.P.
        Bioorg. Med. Chem. Lett. 2006; 16: 1123-1125
        • Itoh K.
        • Huang Z.
        • Liu H.W.
        Org. Lett. 2007; 9: 879-882
        • Carlson E.E.
        • May J.F.
        • Kiessling L.L.
        Chem. Biol. 2006; 13: 825-837
        • Zhang Q.
        • Liu H.
        J. Am. Chem. Soc. 2001; 123: 6756-6766
        • Yuan Y.
        • Bleile D.W.
        • Wen X.
        • Sanders D.A.
        • Itoh K.
        • Liu H.W.
        • Pinto B.M.
        J. Am. Chem. Soc. 2008; 130: 3157-3168
        • Errey J.C.
        • Mann M.C.
        • Fairhurst S.A.
        • Hill L.
        • McNeil M.R.
        • Naismith J.H.
        • Percy J.M.
        • Whitfield C.
        • Field R.A.
        Org. Biomol. Chem. 2009; 7: 1009-1016
        • Yuan Y.
        • Wen X.
        • Sanders D.A.
        • Pinto B.M.
        Biochemistry. 2005; 44: 14080-14089
        • Gruber T.D.
        • Borrok M.J.
        • Westler W.M.
        • Forest K.T.
        • Kiessling L.L.
        J. Mol. Biol. 2009; 391: 327-340
        • Gruber T.D.
        • Westler W.M.
        • Kiessling L.L.
        • Forest K.T.
        Biochemistry. 2009; 48: 9171-9173
        • Feng L.
        • Senchenkova S.N.
        • Yang J.
        • Shashkov A.S.
        • Tao J.
        • Guo H.
        • Cheng J.
        • Ren Y.
        • Knirel Y.A.
        • Reeves P.R.
        • Wang L.
        J. Bacteriol. 2004; 186: 4510-4519
        • Hanniffy O.M.
        • Shashkov A.S.
        • Moran A.P.
        • Prendergast M.M.
        • Senchenkova S.N.
        • Knirel Y.A.
        • Savage A.V.
        Carbohydr. Res. 1999; 319: 124-132
        • Arbatsky N.P.
        • Shashkov A.S.
        • Mamyan S.S.
        • Knirel Y.A.
        • Zych K.
        • Sidorczyk Z.
        Carbohydr. Res. 1998; 310: 85-90
        • St Michael F.
        • Szymanski C.M.
        • Li J.
        • Chan K.H.
        • Khieu N.H.
        • Larocque S.
        • Wakarchuk W.W.
        • Brisson J.R.
        • Monteiro M.A.
        Eur. J. Biochem. 2002; 269: 5119-5136
        • Eppe G.
        • Peltier P.
        • Daniellou R.
        • Nugier-Chauvin C.
        • Ferrières V.
        • Vincent S.P.
        Bioorg. Med. Chem. Lett. 2009; 19: 814-816
        • Wang Q.
        • Ding P.
        • Perepelov A.V.
        • Xu Y.
        • Wang Y.
        • Knirel Y.A.
        • Wang L.
        • Feng L.
        Mol. Microbiol. 2008; 70: 1358-1367
        • Allos B.M.
        Clin. Infect. Dis. 2001; 32: 1201-1206
        • Kaldor J.
        • Speed B.R.
        Br. Med. J. 1984; 288: 1867-1870
        • Delederkremer R.M.
        • Nahmad V.B.
        • Varela O.
        J. Org. Chem. 1994; 59: 690-692
        • Errey J.C.
        • Mukhopadhyay B.
        • Kartha K.P.
        • Field R.A.
        Chem. Commun. 2004; : 2706-2707
        • Miller W.G.
        • Bates A.H.
        • Horn S.T.
        • Brandl M.T.
        • Wachtel M.R.
        • Mandrell R.E.
        Appl. Environ. Microbiol. 2000; 66: 5426-5436
        • Marolda C.L.
        • Welsh J.
        • Dafoe L.
        • Valvano M.A.
        J. Bacteriol. 1990; 172: 3590-3599
        • Carrillo C.D.
        • Taboada E.
        • Nash J.H.
        • Lanthier P.
        • Kelly J.
        • Lau P.C.
        • Verhulp R.
        • Mykytczuk O.
        • Sy J.
        • Findlay W.A.
        • Amoako K.
        • Gomis S.
        • Willson P.
        • Austin J.W.
        • Potter A.
        • Babiuk L.
        • Allan B.
        • Szymanski C.M.
        J. Biol. Chem. 2004; 279: 20327-20338
        • Yao R.
        • Alm R.A.
        • Trust T.J.
        • Guerry P.
        Gene. 1993; 130: 127-130
        • Nothaft H.
        • Liu X.
        • McNally D.J.
        • Li J.
        • Szymanski C.M.
        Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 15019-15024
        • Bacon D.J.
        • Szymanski C.M.
        • Burr D.H.
        • Silver R.P.
        • Alm R.A.
        • Guerry P.
        Mol. Microbiol. 2001; 40: 769-777
        • Tsai C.M.
        • Frasch C.E.
        Anal. Biochem. 1982; 119: 115-119
        • Feldman M.F.
        • Marolda C.L.
        • Monteiro M.A.
        • Perry M.B.
        • Parodi A.J.
        • Valvano M.A.
        J. Biol. Chem. 1999; 274: 35129-35138
        • Marolda C.L.
        • Lahiry P.
        • Vinés E.
        • Saldías S.
        • Valvano M.A.
        Methods Mol. Biol. 2006; 347: 237-252
        • Lambert C.
        • Léonard N.
        • De Bolle X.
        • Depiereux E.
        Bioinformatics. 2002; 18: 1250-1256
        • Morris G.M.
        • Goodsell D.S.
        • Halliday R.S.
        • Huey R.
        • Hart W.E.
        • Belew R.K.
        • Olson A.J.
        J. Comput. Chem. 1998; 19: 1639-1662
        • Bian X.L.
        • Rosas-Acosta G.
        • Wu Y.C.
        • Wilson V.G.
        J. Virol. 2007; 81: 2899-2908
        • Fersht A.
        Julet M.R. Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding. W. H. Freeman & Co., New York1999: 116-117
        • Parkhill 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: 665-668
        • Liu D.
        • Reeves P.R.
        Microbiology. 1994; 140: 49-57
        • Stevenson G.
        • Neal B.
        • Liu D.
        • Hobbs M.
        • Packer N.H.
        • Batley M.
        • Redmond J.W.
        • Lindquist L.
        • Reeves P.
        J. Bacteriol. 1994; 176: 4144-4156
        • Lee R.
        • Monsey D.
        • Weston A.
        • Duncan K.
        • Rithner C.
        • McNeil M.
        Anal. Biochem. 1996; 242: 1-7
        • Karlyshev A.V.
        • Champion O.L.
        • Churcher C.
        • Brisson J.R.
        • Jarrell H.C.
        • Gilbert M.
        • Brochu D.
        • St Michael F.
        • Li J.
        • Wakarchuk W.W.
        • Goodhead I.
        • Sanders M.
        • Stevens K.
        • White B.
        • Parkhill J.
        • Wren B.W.
        • Szymanski C.M.
        Mol. Microbiol. 2005; 55: 90-103
        • Guan S.
        • Clarke A.J.
        • Whitfield C.
        J. Bacteriol. 2001; 183: 3318-3327
        • Bernatchez S.
        • Szymanski C.M.
        • Ishiyama N.
        • Li J.
        • Jarrell H.C.
        • Lau P.C.
        • Berghuis A.M.
        • Young N.M.
        • Wakarchuk W.W.
        J. Biol. Chem. 2005; 280: 4792-4802
        • Cha S.
        Mol. Pharmacol. 1968; 4: 621-629
        • Rose N.L.
        • Palcic M.M.
        • Evans S.V.
        J. Chem. Educ. 2005; 82: 1846-1852
        • Claus H.
        • Stummeyer K.
        • Batzilla J.
        • Mühlenhoff M.
        • Vogel U.
        Mol. Microbiol. 2009; 71: 960-971