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  • Title: Synthesis of the oxaloacetate decarboxylase Na+ pump and its individual subunits in Escherichia coli and analysis of their function.
    Author: Di Berardino M, Dimroth P.
    Journal: Eur J Biochem; 1995 Aug 01; 231(3):790-801. PubMed ID: 7649179.
    Abstract:
    The oadGAB genes encoding the gamma, alpha and beta-subunits of the oxaloacetate decarboxylase Na+ pump in Klebsiella pneumoniae have been cloned on plasmid pSK-GAB and expressed in Escherichia coli. The membranes of the recombinant E. coli clone contained about three times as much catalytically active oxaloacetate decarboxylase (3 mg protein/2 g wet cells) as those of the K. pneumoniae strain from which the genes were derived. The enzyme was solubilised from the membranes with Triton X-100 and purified. Its Na+ transport function was demonstrated after reconstitution into proteoliposomes. Proteoliposomes containing only the membrane-bound subunits beta and gamma (not the peripheral alpha-subunit) were unable to catalyse Na+ translocation in response to a transmembrane Na+ (delta pNa+) or electrical gradient (delta psi). Individual subunits of oxaloacetate decarboxylase and combinations of two subunits were expressed from appropriate derivatives of plasmid pSK-GAB. The hydrophobic subunits beta and beta gamma were membrane-bound as expected. Interestingly, the alpha-subunit was located in the cytoplasm if expressed separately or together with beta, but became membrane-bound if expressed together with gamma. A gamma alpha complex was isolated from such membranes by avidin-Sepharose affinity chromatography. Interactions of the gamma-subunit with the water-soluble alpha-subunit and with the membrane-bound beta-subunit are therefore required to form the oxaloacetate decarboxylase complex. The combinations of separately expressed subunits gamma alpha + beta and beta gamma+alpha were shown to yield the catalytically active enzyme. The alpha or the beta-subunit and the combinations of these subunits with the gamma-subunit were therefore expressed in E. coli in a catalytically competent state. Functional expression of the separate gamma-subunit, however, could not be demonstrated. The alpha-subunit was strongly overexpressed from a pT7-7 derived plasmid, but was only partially biotinylated under these conditions. On coexpression of the birA gene encoding biotin ligase the major part (80-100%) of the overexpressed alpha-subunit was biotinylated. Highly purified alpha-subunit was obtained by fractionated precipitation of the soluble cell fraction with ammonium sulfate. Incubation of the alpha-subunit with oxaloacetate led to a CO2 transfer to its prosthetic biotin group with the formation of stoichiometric amounts of pyruvate. The velocity of the CO2 transfer to the biotin on the alpha-subunit was about three orders of magnitude too low to account for the rate of the overall reaction. The carboxyltransfer reaction was significantly accelerated if the gamma-subunit was additionally present.(ABSTRACT TRUNCATED AT 400 WORDS)
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