SYNTHESIS OF THE OXALOACETATE DECARBOXYLASE NA-COLI AND ANALYSIS OF THEIR FUNCTION( PUMP AND ITS INDIVIDUAL SUBUNITS IN ESCHERICHIA)

The oadGAB genes encoding the gamma, alpha and beta-subunits of the ox aloacetate decarboxylase Na+ pump in Klebsiella pneumoniae have been c loned on plasmid pSK-GAB and expressed in Escherichia coli. The membra nes of the recombinant E. coli clone contained about three times as mu ch catalytically active oxaloacetate decarboxylase (3 mg protein/2 g w et cells) as those of the K. pneumoniae strain from which the genes we re 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 t he membrane-bound subunits beta and gamma (not the peripheral cx-subun it) were unable to catalyse Na+ translocation in response to a transme mbrane Na+ (Delta pNa(+)) or electrical gradient (Delta psi). Individu al subunits of oxaloacetate decarboxylase and combinations of two subu nits were expressed from appropriate derivatives of plasmid pSK-GAB. T he hydrophobic subunits beta and beta gamma were membrane-bound as exp ected. Interestingly, the alpha-subunit was located in the cytoplasm i f expressed separately or together with beta, but became membrane-boun d if expressed together with gamma. A gamma alpha complex was isolated from such membranes by avidin-Sepharose affinity chromatography. Inte ractions of the gamma-subunit with the water-soluble alpha-subunit and with the membrane-bound beta-subunit are therefore required to form t he oxaloacetate decarboxylase complex. The combinations of separately expressed subunits gamma alpha + beta and beta gamma + alpha were show n to yield the catalytically active enzyme. The alpha or the beta-subu nit and the combinations of these subunits with the gamma-subunit were therefore expressed in E. coli in a catalytically competent state. Fu nctional expression of the separate gamma-subunit, however, could not be demonstrated. The alpha-subunit was strongly overexpressed from a p T7-7 derived plasmid, but was only partially biotinylated under these conditions. On coexpression of the birA gene encoding biotin ligase th e major part (80 - 100 %) of the overexpressed alpha-subunit was bioti nylated. Highly purified alpha-subunit was obtained by fractionated pr ecipitation of the soluble cell, fraction with ammonium sulfate. Incub ation of the alpha-subunit with oxaloacetate led to a CO2 transfer to its prosthetic biotin group with the formation of stoichiometric amoun ts 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 f or the rate of the overall reaction. The carboxyltransfer reaction was significantly accelerated if the gamma-subunit was additionally prese nt. This protein was found to contain the Zn2+ cofactor which is belie ved to be directly involved in the catalysis of the carboxyltransfer r eaction.