Cloning, sequencing, and expression in Escherichia coli of OxIT, the oxalate:Formate exchange protein of Oxalobacter formigenes

Abstract

OxIT is the oxalate/formate exchange protein that represents the vectorial component of a proton-motive metabolic cycle in Oxalobacter formigenes. Here we report the cloning and sequencing of OxIT and describe its expression in Escherichia coli. The OxIT amino acid sequence specifies a polytopic hydrophobic protein of 418 residues with a mass of 44,128 daltons. Analysis of hydropathy and consideration of the distribution of charged residues suggests an OxIT secondary structure having 12 transmembrane segments, oriented so that the N and C termini face the cytoplasm. Expression of OxIT in E. coli coincides with appearance of a capacity to carry out the self-exchange of oxalate and the heterologous, electrogenic exchange of oxalate with formate. The unusually high velocity of OxIT-mediated transport is also preserved in E. coli. We conclude that the essential features of OxIT are retained on its expression in E. coli.