Sulphur shuttling across a chaperone during molybdenum cofactor maturation

Arnoux, Pascal; Ruppelt, Christian; Oudouhou, Flore; Lavergne, Jérôme; Siponen, M.I.; Toci, René; Mendel, Ralf R.; Bittner, Florian; Pignol, David; Magalon, Axel; Walburger, Anne

doi:10.1038/ncomms7148

Cited by 42 publications

(59 citation statements)

References 30 publications

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“…Several previous studies showed that GDP can act as a Moco surrogate in Moco-free proteins [68]. The surface signature consistent with the guanine binding site has been also detected in the structure of a D1 homolog [70].…”

Section: Discussionmentioning

confidence: 70%

“…Therefore, D1 aerobic samples could be used for binding experiments to check possible interactions with molecules resembling bis -MGD (Moco type) fragments, such as guanosine diphosphate (GDP). The analysis of available crystal structures of a sulfurtransferase, a chaperone protein for a formate dehydrogenase with bound guanosine diphosphate (GDP) (PDB ID: 4PDE ) [68], and biochemical studies on a DmsD chaperone protein [69,70] suggested that the GDP or GTP can be used to detect interactions between a chaperone and guanosine nucleotide moiety of MGD (Moco type). Therefore, we used GDP as an MGD surrogate and we checked whether D1 aerobic interacts with GDP by thermofluor shift assay (TSA), SEC FPLC and isothermal titration calorimetry (ITC).…”

Section: Resultsmentioning

confidence: 99%

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Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Niedzialkowska

Mrugala

Rugor

et al. 2017

Protein Expression and Purification

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Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor’s insertion into the enzyme and the enzyme’s maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon’s expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

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Section: Discussionmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Niedzialkowska

Mrugala

Rugor

et al. 2017

Protein Expression and Purification

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“…To investigate whether the formate dehydrogenase activity of FDH-H was required for uric acid degradation, urate-degrading activity was examined in an ΔfdhD mutant strain lacking FdhD, which is essential for formate dehydrogenase activity but not for electron-transferring activity. FdhD is a sulfur transferase which transfers sulfur to molybdo-bis-pyranopterin guanine dinucleotide (Mo-bisPGD), an essential cofactor for formate dehydrogenase activity of FDH (30). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification of a Formate-Dependent Uric Acid Degradation Pathway in Escherichia coli

Iwadate

Kato

2019

J Bacteriol

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Purine is a nitrogen-containing compound that is abundant in nature. In organisms that utilize purine as a nitrogen source, purine is converted to uric acid, which is then converted to allantoin. Allantoin is then converted to ammonia. In Escherichia coli, neither urate-degrading activity nor a gene encoding an enzyme homologous to the known urate-degrading enzymes had previously been found.Here, we demonstrate urate-degrading activity in E. coli. We first identified aegA as an E. coli gene involved in oxidative stress tolerance. An examination of gene expression revealed that both aegA and its paralog ygfT are expressed under both microaerobic and anaerobic conditions. The ygfT gene is localized within a chromosomal gene cluster presumably involved in purine catabolism. Accordingly, the expression of ygfT increased in the presence of exogenous uric acid, suggesting that ygfT is involved in urate degradation. Examination of the change of uric acid levels in the growth medium with time revealed urate-degrading activity under microaerobic and anaerobic conditions in the wild-type strain but not in the aegA ygfT double-deletion mutant. Furthermore, AegA-and YgfT-dependent urate-degrading activity was detected only in the presence of formate and formate dehydrogenase H. Collectively, these observations indicate the presence of urate-degrading activity in E. coli that is operational under microaerobic and anaerobic conditions. The activity requires formate, formate dehydrogenase H, and either aegA or ygfT. We also identified other putative genes which are involved not only in formate-dependent but also in formate-independent urate degradation and may function in the regulation or cofactor synthesis in purine catabolism. IMPORTANCE The metabolic pathway of uric acid degradation to date has been elucidated only in aerobic environments and is not understood in anaerobic and microaerobic environments. In the current study, we showed that Escherichia coli, a facultative anaerobic organism, uses uric acid as a sole source of nitrogen under anaerobic and microaerobic conditions. We also showed that formate, formate dehydrogenase H, and either AegA or YgfT are involved in uric acid degradation. We propose that formate may act as an electron donor for a uric acid-degrading enzyme in this bacterium.

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“…One of the isolated mutants had a mutation in fdhE and was deficient in the respiratory formate dehydrogenases N and O only; this mutation was without effect on transcription (Stewart et al, 1991 ). The mutation in another mutant was located in fdhD and had an effect on all three formate dehydrogenases, because FdhD, as was shown recently (Thomé et al, 2012 ; Arnoux et al, 2015 ), is involved in donating a sulfur ligand to the Mo-bis-PGD. It is conceivable that HydN and YsaA could contribute specifically to the cofactor activation of the FDH-H protein, since NAR and FDH-N activities are not impaired in the mutant; however, it would be expected that a more pronounced phenotype of the individual mutants would be observed, making this explanation unlikely.…”

Section: Discussionmentioning

confidence: 70%

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex

Pinske

2018

Front. Microbiol.

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Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

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Sulphur shuttling across a chaperone during molybdenum cofactor maturation

Cited by 42 publications

References 30 publications

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization

Identification of a Formate-Dependent Uric Acid Degradation Pathway in Escherichia coli

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex

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