The Na+-dependent citrate carrier of Klebsiella pneumoniae: high-level expression and site-directed mutagenesis of asparagine-185 and glutamate-194.
The Na+-dependent citrate carrier of Klebsiella pneumoniae (CitS) is a member of the 2-hydroxycarboxylate transporter family. Within the highly conserved helix Vb region, Asn-185 of CitS was mutated to Val and Glu-194 was mutated to Gln. The wild-type and mutant proteins were synthesised in Escherichia coli DH5alpha or C43(DE3) as biotinylated or His-tagged CitS-fusions, respectively. The synthesis and purification procedure yielded 6.5 mg pure CitS per litre culture. The fusion proteins were characterised with E. coli cell suspensions or after reconstitution of the purified enzymes into proteoliposomes. The E194Q mutation had almost no effect on the kinetics of Na+ or citrate transport. In contrast, aberrant citrate transport kinetics were found for the N185V mutant. The apparent K(m) value for the citrate species H-citrate(2-) was increased about nine-fold, whereas the apparent Vmax value and the effect of Na+ on the transport kinetics were comparable to the wild-type. Asn-185 of CitS appears therefore to participate in the binding of H-citrate(2-).
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Institut für Mikrobiologie der Eidgenössischen Technischen Hochschule, ETH-Zentru, Zürich, Switzerland.
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D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D001483:Base Sequence; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D019343:Citric Acid / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D015870:Gene Expression; D005798:Genes, Bacterial; D018698:Glutamic Acid / Q000235:genetics; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012964:Sodium / Q000378:metabolism*; D027981:Symporters
D001426:Bacterial Proteins; D002352:Carrier Proteins; C490118:CitS protein, E coli; C086246:CitS protein, bacteria; D017931:DNA Primers; D029968:Escherichia coli Proteins; D011993:Recombinant Fusion Proteins; D027981:Symporters; D019343:Citric Acid; D018698:Glutamic Acid; D001216:Asparagine; D012964:Sodium