The Na + -dependent citrate carrier of Klebsiella pneumoniae : high-level expression and site-directed mutagenesis of asparagine-185 and glutamate-194

Abstract

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-).

The 2-Hydroxycarboxylate Transporter Family: Physiology, Structure, and Mechanism

“…A complication with the C-terminal BAD is that the C terminus of the CitS protein is located in the periplasm, which results in less efficient biotinylation of the protein, a reaction catalyzed by biotin ligase situated in the cytoplasm (144). Optimization was sought by coexpressing the birA gene, coding for biotin ligase, and by adding biotin to the growth medium (56). The yield of the procedure was further improved by replacing the His 6 tag with a His 10 tag (109), by moving the BAD domain from the C to the N terminus (109), and by using different expression systems, growth conditions, and host strains (56).…”

“…Optimization was sought by coexpressing the birA gene, coding for biotin ligase, and by adding biotin to the growth medium (56). The yield of the procedure was further improved by replacing the His 6 tag with a His 10 tag (109), by moving the BAD domain from the C to the N terminus (109), and by using different expression systems, growth conditions, and host strains (56). These improvements resulted in yields of as much as 6.5 mg of pure protein from 1 liter of culture for the His 10 -tagged CitS protein.…”

“…The copurification of tagged and untagged CitS was taken as evidence for an oligomeric structure of the protein (see "Quartenary Structure" in "STRUCTURE" below) (108). Purified CitS was used for mechanistic studies following reconstitution in proteoliposomes (56,109) and for singlemolecule fluorescence spectroscopy studies (57).…”

“…Later, the solubilized membranes were replaced by purified CitS, resulting in a liposomal system containing essentially one membrane protein. Purified CitS in dodecylmaltoside solution was reconstituted by dilution into a suspension of preformed liposomes made of soybean phosphatidylcholine (56,108,109). Kinetic analysis of citrate transport catalyzed by CitS suggested that the protein was inserted asymmetrically in the liposomal membrane (109).…”

“…Mutation of asparagine to valine in the motif reduced the affinity for citrate by one order of magnitude, suggesting that Asp185 could participate in binding of the substrate (56). Mutation of the glutamate residue Glu194 in the putative pore-loop had little effect on the overall kinetics of CitS (56).…”