title: Highly active and stable oxaloacetate decarboxylase Na⁺ pump complex for structural analysis.
abstract: The oxaloacetate decarboxylase primary Na(+) pump (Oad) produces energy for the surviving of some pathogenic bacteria under anaerobic conditions. Oad composes of three subunits: Oad-α, a biotinylated soluble subunit and catalyzes the decarboxylation of oxaloacetate; Oad-β, a transmembrane subunit and functions as a Na(+) pump; and Oad-γ, a single transmembrane α-helical anchor subunit and assembles Oad-α/β/γ complex. The molecular mechanism of Oad complex coupling the exothermic decarboxylation to generate the Na(+) electrochemical gradient remains unsolved. Our biophysical and biochemical studies suggested that the stoichiometry of Oad complex from Vibrio cholerae composed of α, β, γ in 4:2:2 stoichiometry not that of 4:4:4. The high-resolution structure determination of the Oad complex would reveal the energetic transformation mechanism from the catalytical soluble α subunit to membrane β subunit. Sufficient amount stable, conformational homogenous and active Oad complex with the right stoichiometry is the prerequisite for structural analysis. Here we report an easy and reproducible protocol to obtain high quantity and quality Oad complex protein for structural analysis.
authors.affiliation: The Laboratory of Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland. Electronic address: m.inoue@tagen.tohoku.ac.jp.
authors.forename: Michio
authors.initials: M
authors.lastname: Inoue
authors.affiliation: The Laboratory of Biomolecular Research, Paul Scherrer Institut, 5232 Villigen, Switzerland. Electronic address: xiao.li@psi.ch.
authors.forename: Xiaodan
authors.initials: X
authors.lastname: Li
mesh_terms: D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014734:Vibrio cholerae / Q000235:genetics
chemical_list: D012964:Sodium; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase
keywords: Oxaloacetate decarboxylase primary sodium pump; Vibrio cholerae