New role for the ankyrin repeat revealed by a study of the N-formyltransferase from Providencia alcalifaciens.
ABSTRACT: N-Formylated sugars such as 3,6-dideoxy-3-formamido-d-glucose (Qui3NFo) have been observed on the lipopolysaccharides of various pathogenic bacteria, including Providencia alcalifaciens, a known cause of gastroenteritis. These unusual carbohydrates are synthesized in vivo as dTDP-linked sugars. The biosynthetic pathway for the production of dTDP-Qui3NFo requires five enzymes with the last step catalyzed by an N-formyltransferase that utilizes N(10)-tetrahydrofolate as a cofactor. Here we describe a structural and functional investigation of the P. alcalifaciens N-formyltransferase, hereafter referred to as QdtF. For this analysis, the structure of the dimeric enzyme was determined in the presence of N(5)-formyltetrahydrofolate, a stable cofactor, and dTDP-3,6-dideoxy-3-amino-d-glucose (dTDP-Qui3N) to 1.5 Å resolution. The overall fold of the subunit consists of three regions with the N-terminal and middle motifs followed by an ankyrin repeat domain. Whereas the ankyrin repeat is a common eukaryotic motif involved in protein-protein interactions, reports of its presence in prokaryotic enzymes have been limited. Unexpectedly, this ankyrin repeat houses a second binding pocket for dTDP-Qui3N, which is characterized by extensive interactions between the protein and the ligand. To address the effects of this second binding site on catalysis, a site-directed mutant protein, W305A, was constructed. Kinetic analyses demonstrated that the catalytic activity of the W305A variant was reduced by approximately 7-fold. The structure of the W305A mutant protein in complex with N(5)-formyltetrahydrofolate and dTDP-Qui3N was subsequently determined to 1.5 Å resolution. The electron density map clearly showed that ligand binding had been completely abolished in the auxiliary pocket. The wild-type enzyme was also tested for activity against dTDP-3,6-dideoxy-3-amino-d-galactose (dTDP-Fuc3N) as a substrate. Strikingly, sigmoidal kinetics indicating homotropic allosteric behavior were observed. Although the identity of the ligand that regulates QdtF activity in vivo is at present unknown, our results still provide the first example of an ankyrin repeat functioning in small molecule binding.
Project description:N-formylated sugars are found on the lipopolysaccharides of various pathogenic Gram negative bacteria including Campylobacter jejuni 81116, Francisella tularensis, Providencia alcalifaciens O30, and Providencia alcalifaciens O40. The last step in the biosynthetic pathways for these unusual sugars is catalyzed by N-formyltransferases that utilize N10-formyltetrahydrofolate as the carbon source. The substrates are dTDP-linked amino sugars with the functional groups installed at either the C-3' or C-4' positions of the pyranosyl rings. Here we describe a structural and enzymological investigation of the putative N-formyltransferase, FdtF, from Salmonella enterica O60. In keeping with its proposed role in the organism, the kinetic data reveal that the enzyme is more active with dTDP-3-amino-3,6-dideoxy-d-galactose than with dTDP-3-amino-3,6-dideoxy-d-glucose. The structural data demonstrate that the enzyme contains, in addition to the canonical N-formyltransferase fold, an ankyrin repeat moiety that houses a second dTDP-sugar binding pocket. This is only the second time an ankyrin repeat has been shown to be involved in small molecule binding. The research described herein represents the first structural analysis of a sugar N-formyltransferase that specifically functions on dTDP-3-amino-3,6-dideoxy-d-galactose in vivo and thus adds to our understanding of these intriguing enzymes.
Project description:The O-antigens, which are components of the outer membranes of Gram-negative bacteria, are responsible for the wide species variations seen in nature and are thought to play a role in bacterial virulence. They often contain unusual dideoxysugars such as 3,6-dideoxy-3-formamido-d-glucose (Qui3NFo). Here, we describe a structural and functional investigation of the protein C8J_1081 from Campylobacter jejuni 81116, which is involved in the biosynthesis of Qui3NFo. Specifically, the enzyme, hereafter referred to as WlaRD, catalyzes the N-formylation of dTDP-3,6-dideoxy-3-amino-d-glucose (dTDP-Qui3N) using N(10)-formyltetrahydrofolate as the carbon source. For this investigation, seven X-ray structures of WlaRD, in complexes with various dTDP-linked sugars and cofactors, were determined to resolutions of 1.9 Å or better. One of the models, with bound N(10)-formyltetrahydrofolate and dTDP, represents the first glimpse of an N-formyltransferase with its natural cofactor. Another model contains the reaction products, tetrahydrofolate and dTDP-Qui3NFo. In combination, the structures provide snapshots of the WlaRD active site before and after catalysis. On the basis of these structures, three amino acid residues were targeted for study: Asn 94, His 96, and Asp 132. Mutations of any of these residues resulted in a complete loss of enzymatic activity. Given the position of His 96 in the active site, it can be postulated that it functions as the active site base to remove a proton from the sugar amino group as it attacks the carbonyl carbon of the N-10 formyl group of the cofactor. Enzyme assays demonstrate that WlaRD is also capable of utilizing dTDP-3,6-dideoxy-3-amino-d-galactose (dTDP-Fuc3N) as a substrate, albeit at a much reduced catalytic efficiency.
Project description:The existence of N-formylated sugars in the O-antigens of Gram-negative bacteria has been known since the middle 1980s, but only recently have the biosynthetic pathways for their production been reported. In these pathways, glucose-1-phosphate is first activated by attachment to a dTMP moiety. This step is followed by a dehydration reaction and an amination. The last step in these pathways is catalyzed by N-formyltransferases that utilize N(10) -formyltetrahydrofolate as the carbon source. Here we describe the three-dimensional structure of one of these N-formyltransferases, namely VioF from Providencia alcalifaciens O30. Specifically, this enzyme catalyzes the conversion of dTDP-4-amino-4,6-dideoxyglucose (dTDP-Qui4N) to dTDP-4,6-dideoxy-4-formamido-d-glucose (dTDP-Qui4NFo). For this analysis, the structure of VioF was solved to 1.9 Å resolution in both its apoform and in complex with tetrahydrofolate and dTDP-Qui4N. The crystals used in the investigation belonged to the space group R32 and demonstrated reticular merohedral twinning. The overall catalytic core of the VioF subunit is characterized by a six stranded mixed ?-sheet flanked on one side by three ?-helices and on the other side by mostly random coil. This N-terminal domain is followed by an ?-helix and a ?-hairpin that form the subunit:subunit interface. The active site of the enzyme is shallow and solvent-exposed. Notably, the pyranosyl moiety of dTDP-Qui4N is positioned into the active site by only one hydrogen bond provided by Lys 77. Comparison of the VioF model to that of a previously determined N-formyltransferase suggests that substrate specificity is determined by interactions between the protein and the pyrophosphoryl group of the dTDP-sugar substrate.
Project description:Recent studies have demonstrated that the O-antigens of some pathogenic bacteria such as Brucella abortus, Francisella tularensis, and Campylobacter jejuni contain quite unusual N-formylated sugars (3-formamido-3,6-dideoxy-d-glucose or 4-formamido-4,6-dideoxy-d-glucose). Typically, four enzymes are required for the formation of such sugars: a thymidylyltransferase, a 4,6-dehydratase, a pyridoxal 5'-phosphate or PLP-dependent aminotransferase, and an N-formyltransferase. To date, there have been no published reports of N-formylated sugars associated with Mycobacterium tuberculosis. A recent investigation from our laboratories, however, has demonstrated that one gene product from M. tuberculosis, Rv3404c, functions as a sugar N-formyltransferase. Given that M. tuberculosis produces l-rhamnose, both a thymidylyltransferase (Rv0334) and a 4,6-dehydratase (Rv3464) required for its formation have been identified. Thus, there is one remaining enzyme needed for the production of an N-formylated sugar in M. tuberculosis, namely a PLP-dependent aminotransferase. Here we demonstrate that the M. tuberculosis rv3402c gene encodes such an enzyme. Our data prove that M. tuberculosis contains all of the enzymatic activities required for the formation of dTDP-4-formamido-4,6-dideoxy-d-glucose. Indeed, the rv3402c gene product likely contributes to virulence or persistence during infection, though its temporal expression and location remain to be determined.
Project description:Campylobacter jejuni is a Gram-negative bacterium that represents a leading cause of human gastroenteritis worldwide. Of particular concern is the link between C. jejuni infections and the subsequent development of Guillain-Barré syndrome, an acquired autoimmune disorder leading to paralysis. All Gram-negative bacteria contain complex glycoconjugates anchored to their outer membranes, but in most strains of C. jejuni, this lipoglycan lacks the O-antigen repeating units. Recent mass spectrometry analyses indicate that the C. jejuni 81116 (Penner serotype HS:6) lipoglycan contains two dideoxyhexosamine residues, and enzymological assay data show that this bacterial strain can synthesize both dTDP-3-acetamido-3,6-dideoxy-d-glucose and dTDP-3-acetamido-3,6-dideoxy-d-galactose. The focus of this investigation is on WlaRG from C. jejuni, which plays a key role in the production of these unusual sugars by functioning as a pyridoxal 5'-phosphate dependent aminotransferase. Here, we describe the first three-dimensional structures of the enzyme in various complexes determined to resolutions of 1.7 Å or higher. Of particular significance are the external aldimine structures of WlaRG solved in the presence of either dTDP-3-amino-3,6-dideoxy-d-galactose or dTDP-3-amino-3,6-dideoxy-d-glucose. These models highlight the manner in which WlaRG can accommodate sugars with differing stereochemistries about their C-4' carbon positions. In addition, we present a corrected structure of WbpE, a related sugar aminotransferase from Pseudomonas aeruginosa, solved to 1.3 Å resolution.
Project description:N-formylated sugars have been observed on the O-antigens of such pathogenic Gram-negative bacteria as Campylobacter jejuni and Francisella tularensis. Until recently, however, little was known regarding the overall molecular architectures of the N-formyltransferases that are required for the biosynthesis of these unusual sugars. Here we demonstrate that the protein encoded by the wbtj gene from F. tularensis is an N-formyltransferase that functions on dTDP-4-amino-4,6-dideoxy-d-glucose as its substrate. The enzyme, hereafter referred to as WbtJ, demonstrates a strict requirement for N(10) -formyltetrahydrofolate as its carbon source. In addition to the kinetic analysis, the three-dimensional structure of the enzyme was solved in the presence of dTDP-sugar ligands to a nominal resolution of 2.1 Å. Each subunit of the dimeric enzyme is dominated by a "core" domain defined by Met 1 to Ser 185. This core motif harbors the active site residues. Following the core domain, the last 56 residues fold into two ?-helices and a ?-hairpin motif. The hairpin motif is responsible primarily for the subunit:subunit interface, which is characterized by a rather hydrophobic pocket. From the study presented here, it is now known that WbtJ functions on C-4' amino sugars. Another enzyme recently investigated in the laboratory, WlaRD, formylates only C-3' amino sugars. Strikingly, the quaternary structures of WbtJ and WlaRD are remarkably different. In addition, there are several significant variations in the side chains that line their active site pockets, which may be important for substrate specificity. Details concerning the kinetic and structural properties of WbtJ are presented.
Project description:Pantoea ananatis is a Gram-negative bacterium first recognized in 1928 as the causative agent of pineapple rot in the Philippines. Since then various strains of the organism have been implicated in the devastation of agriculturally important crops. Some strains, however, have been shown to function as non-pathogenic plant growth promoting organisms. To date, the factors that determine pathogenicity or lack thereof between the various strains are not well understood. All P. ananatis strains contain lipopolysaccharides, which differ with respect to the identities of their associated sugars. Given our research interest on the presence of the unusual sugar, 4-formamido-4,6-dideoxy-d-glucose, found on the lipopolysaccharides of Campylobacter jejuni and Francisella tularensis, we were curious as to whether other bacteria have the appropriate biosynthetic machinery to produce these unique carbohydrates. Four enzymes are typically required for their biosynthesis: a thymidylyltransferase, a 4,6-dehydratase, an aminotransferase, and an N-formyltransferase. Here, we report that the gene SAMN03097714_1080 from the P. ananatis strain NFR11 does, indeed, encode for an N-formyltransferase, hereafter referred to as PA1080c. Our kinetic analysis demonstrates that PA1080c displays classical Michaelis-Menten kinetics with dTDP-4-amino-4,6-dideoxy-d-glucose as the substrate and N10 -formyltetrahydrofolate as the carbon source. In addition, the X-ray structure of PA1080c, determined to 1.7 Å resolution, shows that the enzyme adopts the molecular architecture observed for other sugar N-formyltransferases. Analysis of the P. ananatis NFR11 genome suggests that the three other enzymes necessary for N-formylated sugar biosynthesis are also present. Intriguingly, those strains of P. ananatis that are non-pathogenic apparently do not contain these genes.
Project description:Derivatives of 3-amino-3,6-dideoxyhexoses are widespread in Nature. They are part of the repeating units of lipopolysaccharide O-antigens, of the glycan moiety of S-layer (bacterial cell surface layer) glycoproteins and also of many antibiotics. In the present study, we focused on the elucidation of the biosynthesis pathway of dTDP-alpha-D-Quip3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-glucose) from the Gram-positive, anaerobic, thermophilic organism Thermoanaerobacterium thermosaccharolyticum E207-71, which carries Quip3NAc in its S-layer glycan. The biosynthesis of dTDP-alpha-D-Quip3NAc involves five enzymes, namely a transferase, a dehydratase, an isomerase, a transaminase and a transacetylase, and follows a pathway similar to that of dTDP-alpha-D-Fucp3NAc (dTDP-3-acetamido-3,6-dideoxy-alpha-D-galactose) biosynthesis in Aneurinibacillus thermoaerophilus L420-91(T). The ORFs (open reading frames) of interest were cloned, overexpressed in Escherichia coli and purified. To elucidate the enzymatic cascade, the different products were purified by HPLC and characterized by NMR spectroscopy. The initiating reactions catalysed by the glucose-1-phosphate thymidylyltransferase RmlA and the dTDP-D-glucose-4,6-dehydratase RmlB are well established. The subsequent isomerase was shown to be capable of forming a dTDP-3-oxo-6-deoxy-D-glucose intermediate from the RmlB product dTDP-4-oxo-6-deoxy-D-glucose, whereas the isomerase involved in the dTDP-alpha-D-Fucp3NAc pathway synthesizes dTDP-3-oxo-6-deoxy-D-galactose. The subsequent reaction steps of either pathway involve a transaminase and a transacetylase, leading to the specific production of nucleotide-activated 3-acetamido-3,6-dideoxy-alpha-D-glucose and 3-acetamido-3,6-dideoxy-alpha-D-galactose respectively. Sequence comparison of the ORFs responsible for the biosynthesis of dTDP-alpha-D-Quip3NAc revealed homologues in Gram-negative as well as in antibiotic-producing Gram-positive bacteria. There is strong evidence that the elucidated biosynthesis pathway may also be valid for LPS (lipopolysaccharide) O-antigen structures and antibiotic precursors.
Project description:The Gram-negative bacterium Campylobacter jejuni 81116 (Penner serotype HS:6) has a class E lipooligosaccharide (LOS) biosynthesis locus containing 19 genes, which encode for 11 putative glycosyltransferases, 1 lipid A acyltransferase and 7 enzymes thought to be involved in the biosynthesis of dideoxyhexosamine (ddHexN) moieties. Although the LOS outer core structure of C. jejuni 81116 is still unknown, recent mass spectrometry analyses suggest that it contains acetylated forms of two ddHexN residues. For this investigation, five of the genes encoding enzymes reportedly involved in the biosyntheses of these sugar residues were examined, rmlA, rmlB, wlaRA, wlaRB and wlaRG. Specifically, these genes were cloned and expressed in Escherichia coli, and the corresponding enzymes were purified and tested for biochemical activity. Here we present data demonstrating that RmlA functions as a glucose-1-phosphate thymidylyltransferase and that RmlB is a thymidine diphosphate (dTDP)-glucose 4,6-dehydratase. We also show, through nuclear magnetic resonance spectroscopy and mass spectrometry analyses, that WlaRG, when utilized in coupled assays with either WlaRA or WlaRB and dTDP-4-keto-6-deoxyglucose, results in the production of either dTDP-3-amino-3,6-dideoxy-d-galactose (dTDP-Fuc3N) or dTDP-3-amino-3,6-dideoxy-d-glucose (dTDP-Qui3N), respectively. In addition, the X-ray crystallographic structures of the 3,4-ketoisomerases, WlaRA and WlaRB, were determined to 2.14 and 2.0 Å resolutions, respectively. Taken together, the data reported herein demonstrate that C. jejuni 81116 utilizes five enzymes to synthesize dTDP-Fuc3N or dTDP-Qui3N and that WlaRG, an aminotransferase, can function on sugars with differing stereochemistry about their C-4' carbons. Importantly, the data reveal that C. jejuni 81116 has the ability to synthesize two isomeric ddHexN forms.
Project description:The causative agent of tuberculosis, Mycobacterium tuberculosis, is a bacterium with a complex cell wall and a complicated life cycle. The genome of M. tuberculosis contains well over 4000 genes thought to encode proteins. One of these codes for a putative enzyme referred to as Rv3404c, which has attracted research attention as a potential virulence factor for over 12 years. Here we demonstrate that Rv3404c functions as a sugar N-formyltransferase that converts dTDP-4-amino-4,6-dideoxyglucose into dTDP-4-formamido-4,6-dideoxyglucose using N10-formyltetrahydrofolate as the carbon source. Kinetic analyses demonstrate that Rv3404c displays a significant catalytic efficiency of 1.1 × 104 M-1 s-1. In addition, we report the X-ray structure of a ternary complex of Rv3404c solved in the presence of N5-formyltetrahydrofolate and dTDP-4-amino-4,6-dideoxyglucose. The final model of Rv3404c was refined to an overall R-factor of 16.8% at 1.6 Å resolution. The results described herein are especially intriguing given that there have been no published reports of N-formylated sugars associated with M. tuberculosis. The data thus provide a new avenue of research into this fascinating, yet deadly, organism that apparently has been associated with human infection since ancient times.