Abstract
The bacterial Sox (sulfur oxidizing) system allows the utilization of inorganic sulfur compounds in energy metabolism. Central to this process is the SoxYZ complex that carries the pathway intermediates on a cysteine residue near the C terminus of SoxY. Crystal structures have been determined for Paracoccus pantotrophus SoxYZ with the carrier cysteine in the underivatized state, conjugated to the polysulfide mimic beta-mercaptoethanol, and as the sulfonate adduct pathway intermediate. The carrier cysteine is located on a peptide swinging arm and is bracketed on either side by diglycine dipeptides acting as molecular universal joints. This structure provides a novel solution to the requirement that the cysteine-bound intermediates be able to access and orient themselves within the active sites of multiple partner enzymes. Adjacent to the swinging arm there is a conserved, deep, apolar pocket into which the beta-mercaptoethanol adduct extends. This pocket would be well suited to a role in protecting labile pathway intermediates from adventitious reactions.
Highlights
The Sox4 system appears to be the most widely distributed sulfur oxidation pathway and is found in both photosynthetic and non-photosynthetic sulfur-oxidizing
The Crystal Structure of SoxYZ—Recombinant hexahistidine-tagged P. pantotrophus SoxYZ complex was expressed in the cytoplasm of E. coli and purified in the presence of dithiothreitol
The multiple structures of SoxYZ reported here reveal that the carrier cysteine residue is located on a swinging arm at the C terminus of the SoxY subunit
Summary
Preparation of Plasmids for Heterologous Coexpression of SoxYZ— A plasmid suitable for heterologous co-expression of P. pantotrophus LMD82.5T SoxYZ proteins in Escherichia coli was constructed. Crystals of native P. pantotrophus SoxYZ were grown under similar conditions to those found for the -mercaptoethanol derivative and were cryoprotected by addition of 15% (v/v) ethylene glycol. They were found to be essentially isomorphous with those of the selenomethionine protein and the structure was solved by molecular replacement using a monomer of selenomethionyl -mercaptoethanol SoxYZ as search model. The protein concentration and crystallization condition used were similar to those employed for native SoxYZ and for the -mercaptoethanol derivative For this form of the protein, the crystals were of space group P1 with four copies of the heterodimer in the asymmetric unit.
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