Dataset Information

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30.

ABSTRACT: 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 β-sheet flanked on one side by three α-helices and on the other side by mostly random coil. This N-terminal domain is followed by an α-helix and a β-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.

SUBMITTER: Genthe NA

PROVIDER: S-EPMC4456110 | biostudies-literature | 2015 Jun

REPOSITORIES: biostudies-literature

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Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30.

Genthe Nicholas A NA Thoden James B JB Benning Matthew M MM Holden Hazel M HM

Protein science : a publication of the Protein Society 20150402 6

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 ...[more]

PMID: 25752909

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Project description: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:Cytolethal distending toxins (CDTs), which block eukaryotic cell proliferation by acting as inhibitory cyclomodulins, are produced by diverse groups of Gram-negative bacteria. Active CDT is composed of three polypeptides--CdtA, CdtB, and CdtC--encoded by the genes cdtA, cdtB, and cdtC, respectively. We developed a PCR-restriction fragment length polymorphism assay for the detection and differentiation of five alleles of cdtB (Cdt-I through Cdt-V) in Escherichia coli and used the assay to investigate the prevalence and characteristic of CDT-producing E. coli in children with diarrhea (A. Hinenoya et al., Microbiol. Immunol. 53:206-215, 2009). In these assays, two untypable cdtB genes were detected and the organisms harboring the cdtB gene were identified as Providencia alcalifaciens (strains AH-31 and AS-1). Nucleotide sequence analysis of the cdt gene cluster revealed that the cdtA, cdtB, and cdtC genes of P. alcalifaciens are of 750, 810, and 549 bp, respectively. To understand the possible horizontal transfer of the cdt genes among closely related species, the presence of cdt genes was screened in various Providencia spp. by colony hybridization assay, and the cdt gene cluster was found in only limited strains of P. alcalifaciens. Genome walking revealed that the cdt gene cluster of P. alcalifaciens is located adjacent to a putative transposase gene, suggesting the locus might be horizontally transferable. Interestingly, the CDT of P. alcalifaciens (PaCDT) showed some homology with the CDT of Shigella boydii. Whereas filter-sterilized lysates of strains AH-31 and AS-1 showed distention of CHO but not of HeLa cells, E. coli CDT-I exhibited distention of both cells. This activity of PaCDT was confirmed by generating recombinant PaCDT protein, which could also be neutralized by rabbit anti-PaCdtB antibody. Furthermore, recombinant PaCDT was found to induce G(2)/M cell cycle arrest and phosphorylation of host histone H2AX, a sensitive marker of DNA double-strand breaks. To our knowledge, this is the first report showing that certain clinical P. alcalifaciens strains could produce variants of the CDTs compared.

Project description:IntroductionAcute haemorrhagic diarrhoea syndrome (AHDS) in dogs is a condition of unknown aetiology. Providencia alcalifaciens is suspected to play a role in the disease as it was commonly found in dogs suffering from AHDS during a Norwegian outbreak in 2019. The role of this bacterium as a constituent of the canine gut microbiota is unknown, hence this study set out to investigate its occurrence in healthy dogs using metagenomics.Materials and methodsTo decrease the likelihood of false detection, we established a metagenomic threshold for P. alcalifaciens by spiking culture-negative stool samples with a range of bacterial dilutions and analysing these by qPCR and shotgun metagenomics. The detection limit for P. alcalifaciens was determined and used to establish a metagenomic threshold. The threshold was validated on naturally contaminated faecal samples with known cultivation status for P. alcalifaciens. Finally, the metagenomic threshold was used to determine the occurrence of P. alcalifaciens in shotgun metagenomic datasets from canine faecal samples (n=362) collected in the HUNT One Health project.ResultsThe metagenomic assay and qPCR had a detection limit of 1.1x103 CFU P. alcalifaciens per faecal sample, which corresponded to a Cq value of 31.4 and 569 unique k-mer counts by shotgun metagenomics. Applying this metagenomic threshold to 362 faecal metagenomic datasets from healthy dogs, P. alcalifaciens was found in only 1.1% (95% CI [0.0, 6.8]) of the samples, and then in low relative abundances (median: 0.04%; range: 0.00 to 0.81%). The sensitivity of the qPCR and shotgun metagenomics assay was low, as only 40% of culture-positive samples were also positive by qPCR and metagenomics.DiscussionUsing our detection limit, the occurrence of P. alcalifaciens in faecal samples from healthy dogs was low. Given the low sensitivity of the metagenomic assay, these results do not rule out a significantly higher occurrence of this bacterium at a lower abundance.

Dataset Information

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30.

Publications

Molecular structure of an N-formyltransferase from Providencia alcalifaciens O30.

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