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Title: An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III)

Authors:
; ; ; ; ; ; ; ;  [1];  [2];  [2]
  1. ULC
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1243113
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Inorganic Biochemistry; Journal Volume: 154; Journal Issue: 01, 2016
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Ouellet, Yannick H., Ndiaye, Cheikh Tidiane, Gagné, Stéphane M., Sebilo, Anne, Suits, Michael D.L., Jubinville, Éric, Jia, Zongchao, Ivancich, Anabella, Couture, Manon, Queens), and CNRS-UMR). An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III). United States: N. p., 2016. Web. doi:10.1016/j.jinorgbio.2015.11.002.
Ouellet, Yannick H., Ndiaye, Cheikh Tidiane, Gagné, Stéphane M., Sebilo, Anne, Suits, Michael D.L., Jubinville, Éric, Jia, Zongchao, Ivancich, Anabella, Couture, Manon, Queens), & CNRS-UMR). An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III). United States. doi:10.1016/j.jinorgbio.2015.11.002.
Ouellet, Yannick H., Ndiaye, Cheikh Tidiane, Gagné, Stéphane M., Sebilo, Anne, Suits, Michael D.L., Jubinville, Éric, Jia, Zongchao, Ivancich, Anabella, Couture, Manon, Queens), and CNRS-UMR). Wed . "An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III)". United States. doi:10.1016/j.jinorgbio.2015.11.002.
@article{osti_1243113,
title = {An alternative reaction for heme degradation catalyzed by the Escherichia coli O157:H7 ChuS protein: Release of hematinic acid, tripyrrole and Fe(III)},
author = {Ouellet, Yannick H. and Ndiaye, Cheikh Tidiane and Gagné, Stéphane M. and Sebilo, Anne and Suits, Michael D.L. and Jubinville, Éric and Jia, Zongchao and Ivancich, Anabella and Couture, Manon and Queens) and CNRS-UMR)},
abstractNote = {},
doi = {10.1016/j.jinorgbio.2015.11.002},
journal = {Journal of Inorganic Biochemistry},
number = 01, 2016,
volume = 154,
place = {United States},
year = {Wed Jul 06 00:00:00 EDT 2016},
month = {Wed Jul 06 00:00:00 EDT 2016}
}
  • Heme oxygenases catalyze the oxidation of heme to biliverdin, CO, and free iron. For pathogenic microorganisms, heme uptake and degradation are critical mechanisms for iron acquisition that enable multiplication and survival within hosts they invade. Here we report the first crystal structure of the pathogenic Escherichia coli O157:H7 heme oxygenase ChuS in complex with heme at 1.45 {angstrom} resolution. When compared with other heme oxygenases, ChuS has a unique fold, including structural repeats and a {beta}-sheet core. Not surprisingly, the mode of heme coordination by ChuS is also distinct, whereby heme is largely stabilized by residues from the C-terminal domain,more » assisted by a distant arginine from the N-terminal domain. Upon heme binding, there is no large conformational change beyond the fine tuning of a key histidine (His-193) residue. Most intriguingly, in contrast to other heme oxygenases, the propionic side chains of heme are orientated toward the protein core, exposing the {alpha}-meso carbon position where O{sub 2} is added during heme degradation. This unique orientation may facilitate presentation to an electron donor, explaining the significantly reduced concentration of ascorbic acid needed for the reaction. Based on the ChuS-heme structure, we converted the histidine residue responsible for axial coordination of the heme group to an asparagine residue (H193N), as well as converting a second histidine to an alanine residue (H73A) for comparison purposes. We employed spectral analysis and CO measurement by gas chromatography to analyze catalysis by ChuS, H193N, and H73A, demonstrating that His-193 is the key residue for the heme-degrading activity of ChuS.« less
  • Crystals of Stx2 were grown in the presence of adenosine and adenine. In both cases, the resulting electron density showed only adenine bound at the active site of the A subunit, proving that the holotoxin is an active N-glycosidase. Stx2 is a protein toxin whose catalytic subunit acts as an N-glycosidase to depurinate a specific adenine base from 28S rRNA. In the holotoxin, the catalytic portion, A1, is linked to the rest of the A subunit, A2, and A2 interacts with the pentameric ring formed by the five B subunits. In order to test whether the holotoxin is active asmore » an N-glycosidase, Stx2 was crystallized in the presence of adenosine and adenine. The crystals diffracted to ∼1.8 Å and showed clear electron density for adenine in the active site. Adenosine had been cleaved, proving that Stx2 is an active N-glycosidase. While the holotoxin is active against small substrates, it would be expected that the B subunits would interfere with the binding of the 28S rRNA.« less
  • Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFUs ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less
  • There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9-O-sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into twomore » subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.« less
  • Rapid detection and characterization of food borne pathogens such as Escherichia coli O157:H7 is crucial for epidemiological investigations and food safety surveillance. As an alternative to conventional technologies, we examined the sensitivity and specificity of nucleic acid microarrays for detecting and genotyping E. coli O157:H7. The array was composed of oligonucleotide probes (25-30 mer) complementary to four virulence loci (intimin, Shiga-like toxins I and II, and hemolysin A). Target DNA was amplified from whole cells or from purified DNA via single or multiplexed polymerase chain reaction (PCR), and PCR products were hybridized to the array without further modification or purification.more » The array was 32-fold more sensitive than gel electrophoresis and capable of detecting amplification products from < 1 cell equivalent of genomic DNA (1 fg). Immunomagnetic capture, PCR and a microarray were subsequently used to detect 55 CFU ml-1 (E. coli O157:H7) from chicken rinsate without the aid of pre-enrichment. Four isolates of E. coli O157:H7 and one isolate of O91:H2, for which genotypic data were available, were unambiguously genotyped with this array. Glass based microarrays are relatively simple to construct and provide a rapid and sensitive means to detect multiplexed PCR products and the system is amenable to automation.« less