Synthesis of the oxaloacetate decarboxylase Na+ pump and its individual subunits in Escherichia coli and analysis of their function.
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)
Di Berardino
M
M
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Dimroth
P
P
D001483:Base Sequence; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D017136:Ion Transport; D007711:Klebsiella pneumoniae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015032:Zinc / Q000032:analysis
D017931:DNA Primers; D011994:Recombinant Proteins; D012964:Sodium; D002262:Carboxy-Lyases; C021916:oxaloacetate decarboxylase; D000254:Sodium-Potassium-Exchanging ATPase; D015032:Zinc