Abstract
Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. FDHs activity in Escherichia coli strictly requires the sulphurtransferase EcFdhD, which likely transfers sulphur from IscS to the molybdenum cofactor (Mo-bisPGD) of FDHs. Here we show that EcFdhD binds Mo-bisPGD in vivo and has submicromolar affinity for GDP-used as a surrogate of the molybdenum cofactor's nucleotide moieties. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer. These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. On the basis of structure-guided mutagenesis, we propose a model for the sulphuration mechanism of Mo-bisPGD where the sulphur atom shuttles across the chaperone dimer.
Highlights
Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel
To further characterize the purified EcFdhD protein, the molybdenum content and the stable oxidation product derived from the cofactor were quantified by inductively coupled plasma mass spectrometry (ICP-MS) and high-performance liquid chromatography (HPLC) detection, respectively (Supplementary Table 1)
The copurification of EcFdhD with molybdenum cofactor to a similar extent as measured in R. capsulatus FdsC15 confirms the ability of EcFdhD to protect the unstable and air-sensitive cofactor, suggesting that FdhD plays the role of a chaperone
Summary
Formate dehydrogenases (FDHs) are of interest as they are natural catalysts that sequester atmospheric CO2, generating reduced carbon compounds with possible uses as fuel. The crystal structure of EcFdhD in complex with GDP shows two symmetrical binding sites located on the same face of the dimer These binding sites are connected via a tunnel-like cavity to the opposite face of the dimer where two dynamic loops, each harbouring two functionally important cysteine residues, are present. As nucleotides are chemical moieties of the Mo-bisPGD structure[14], it has been hypothesized that EcFdhD could bind and sulphurate the cofactor before its insertion into FDHs11. In this model, sulphur is transported across the core of EcFdhD from IscS to the molybdenum cofactor, thanks to the presence of a dynamic cysteine loop that reaches across the dimer
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