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Title: Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation

Abstract

Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO–rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rathermore » than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.« less

Authors:
; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1397309
Resource Type:
Journal Article
Resource Relation:
Journal Name: Journal of Biological Chemistry; Journal Volume: 292; Journal Issue: 34
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Motl, Nicole, Skiba, Meredith A., Kabil, Omer, Smith, Janet L., and Banerjee, Ruma. Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation. United States: N. p., 2017. Web. doi:10.1074/jbc.M117.790170.
Motl, Nicole, Skiba, Meredith A., Kabil, Omer, Smith, Janet L., & Banerjee, Ruma. Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation. United States. doi:10.1074/jbc.M117.790170.
Motl, Nicole, Skiba, Meredith A., Kabil, Omer, Smith, Janet L., and Banerjee, Ruma. Thu . "Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation". United States. doi:10.1074/jbc.M117.790170.
@article{osti_1397309,
title = {Structural and biochemical analyses indicate that a bacterial persulfide dioxygenase–rhodanese fusion protein functions in sulfur assimilation},
author = {Motl, Nicole and Skiba, Meredith A. and Kabil, Omer and Smith, Janet L. and Banerjee, Ruma},
abstractNote = {Hydrogen sulfide (H2S) is a signaling molecule that is toxic at elevated concentrations. In eukaryotes, it is cleared via a mitochondrial sulfide oxidation pathway, which comprises sulfide quinone oxidoreductase, persulfide dioxygenase (PDO), rhodanese, and sulfite oxidase and converts H2S to thiosulfate and sulfate. Natural fusions between the non-heme iron containing PDO and rhodanese, a thiol sulfurtransferase, exist in some bacteria. However, little is known about the role of the PDO–rhodanese fusion (PRF) proteins in sulfur metabolism. Herein, we report the kinetic properties and the crystal structure of a PRF from the Gram-negative endophytic bacterium Burkholderia phytofirmans. The crystal structures of wild-type PRF and a sulfurtransferase-inactivated C314S mutant with and without glutathione were determined at 1.8, 2.4, and 2.7 Å resolution, respectively. We found that the two active sites are distant and do not show evidence of direct communication. The B. phytofirmans PRF exhibited robust PDO activity and preferentially catalyzed sulfur transfer in the direction of thiosulfate to sulfite and glutathione persulfide; sulfur transfer in the reverse direction was detectable only under limited turnover conditions. Together with the kinetic data, our bioinformatics analysis reveals that B. phytofirmans PRF is poised to metabolize thiosulfate to sulfite in a sulfur assimilation pathway rather than in sulfide stress response as seen, for example, with the Staphylococcus aureus PRF or sulfide oxidation and disposal as observed with the homologous mammalian proteins.},
doi = {10.1074/jbc.M117.790170},
journal = {Journal of Biological Chemistry},
number = 34,
volume = 292,
place = {United States},
year = {Thu Jul 06 00:00:00 EDT 2017},
month = {Thu Jul 06 00:00:00 EDT 2017}
}
  • Highlights: • Three dimensional solution NMR structure of YgaP rhodanese domain. • Function validation of YgaP rhodanese domain to substrate Na{sub 2}S{sub 2}O{sub 3}. • Fast exchange between the intact and persulfide-intermediate rhodanese domain. - Abstract: Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical rhodanese domain fold was observed, as expected frommore » the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional {sup 1}H–{sup 15}N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.« less
  • Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO 2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. Wemore » also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. Finally, the new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.« less
  • Annually, half of all plant-derived carbon is added to soil where it is microbially respired to CO 2. However, understanding of the microbiology of this process is limited because most culture-independent methods cannot link metabolic processes to the organisms present, and this link to causative agents is necessary to predict the results of perturbations on the system. We collected soil samples at two sub-root depths (10–20 cm and 30–40 cm) before and after a rainfall-driven nutrient perturbation event in a Northern California grassland that experiences a Mediterranean climate. From ten samples, we reconstructed 198 metagenome-assembled genomes that represent all major phylotypes. Wemore » also quantified 6,835 proteins and 175 metabolites and showed that after the rain event the concentrations of many sugars and amino acids approach zero at the base of the soil profile. Unexpectedly, the genomes of novel members of the Gemmatimonadetes and Candidate Phylum Rokubacteria phyla encode pathways for methylotrophy. We infer that these abundant organisms contribute substantially to carbon turnover in the soil, given that methylotrophy proteins were among the most abundant proteins in the proteome. Previously undescribed Bathyarchaeota and Thermoplasmatales archaea are abundant in deeper soil horizons and are inferred to contribute appreciably to aromatic amino acid degradation. Many of the other bacteria appear to breakdown other components of plant biomass, as evidenced by the prevalence of various sugar and amino acid transporters and corresponding hydrolyzing machinery in the proteome. Overall, our work provides organism-resolved insight into the spatial distribution of bacteria and archaea whose activities combine to degrade plant-derived organics, limiting the transport of methanol, amino acids and sugars into underlying weathered rock. The new insights into the soil carbon cycle during an intense period of carbon turnover, including biogeochemical roles to previously little known soil microbes, were made possible via the combination of metagenomics, proteomics, and metabolomics.« less