title: Identification, purification, and reconstitution of OxlT, the oxalate: formate antiport protein of Oxalobacter formigenes.
abstract: We had proposed earlier that the anaerobe Oxalobacter formigenes sustains a proton-motive force by exploiting a secondary carrier rather than a primary proton pump. In this view, a carrier protein would catalyze the exchange of extracellular oxalate, a divalent anion, and intracellular formate, the monovalent product of oxalate decarboxylation. Such an electrogenic exchange develops an internally negative membrane potential, and since the decarboxylation reaction consumes an internal proton, the combined activity of the carrier and the soluble decarboxylase would constitute an "indirect" proton pump with a stoichiometry of 1H+ per turnover. This model is now verified by identification and purification of OxlT, the protein responsible for the anion exchange reaction. Membranes of O. formigenes were solubilized at pH 7 with 1.25% octyl glucoside in 20 mM 3-(N-morpholino)propanesulfonic acid/K, in the presence of 0.4% Escherichia coli phospholipids and with 20% glucerol present as the osmolyte stabilant. Rapid methods for reconstitution were developed to monitor the distribution of OxlT during biochemical fractionation, allowing its purification by sequential anion and cation exchange chromatography. OxlT proved to be a single hydrophobic polypeptide, of 38 kDa mobility during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, with a turnover number estimated as at least 1000/s. The properties of OxlT point to an indirect proton pump as the mechanism by which a proton-motive force arises in O. formigenes, and one may reasonably argue that indirect proton pumps take part in bacterial events such as acetogenesis, malolactate fermentation, and perhaps methanogenesis.
authors.affiliation: Department of Physiology, Johns Hopkins University Medical School, Baltimore, Maryland 21205.
authors.forename: Z S
authors.initials: ZS
authors.lastname: Ruan
authors.forename: V
authors.initials: V
authors.lastname: Anantharam
authors.forename: I T
authors.initials: IT
authors.lastname: Crawford
authors.forename: S V
authors.initials: SV
authors.lastname: Ambudkar
authors.forename: S Y
authors.initials: SY
authors.lastname: Rhee
authors.forename: M J
authors.initials: MJ
authors.lastname: Allison
authors.forename: P C
authors.initials: PC
authors.lastname: Maloney
mesh_terms: D001426:Bacterial Proteins / Q000302:isolation & purification / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates / Q000378:metabolism*; D006089:Gram-Negative Anaerobic Bacteria / Q000737:chemistry / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D002352:Carrier Proteins; D005561:Formates; D010070:Oxalates