Journal of Bacteriology, October 2002, p . 5491-5494, Vol . 184, No . 19

Characterization of Amino Acid Substitutions in KdpA, the K⁺-Binding and -Translocating Subunit of the KdpFABC Complex of Escherichia coli

Martin van der Laan, Michael Gaßel, and Karlheinz Altendorf^*

Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, D-49069 Osnabrück, Germany

Received 19 February 2002/ Accepted 9 July 2002

Construction of site-directed mutants. The E . coli strains and plasmids used in this study are listed in Table 1 . A prerequisite for site-directed mutagenesis was plasmid pSM2, which was constructed from pSM1 (10) (Table 1) by introducing an additional KpnI site (silent mutation) into the kdpA gene . Further mutagenesis was performed by one-step PCR, using plasmid pSM2 as template . Mutagenized plasmids were designated according to their corresponding mutations; e.g., the pSM2 derivative carrying the mutation in kdpA that encoded the G232A change was named pSM-kdpA:G232A .

 
In vivo characterization of the mutants. In order to test the effects of the different mutations on the K⁺ affinity of the KdpFABC complexes in vivo, strain TKW3205, which contains no functional K⁺ uptake system, was transformed with the different plasmids listed in Table 1 . Cells were grown as described previously (19) . Ampicillin-resistant single colonies were transferred to minimal medium agar plates containing 0 to 115 mM KCl (K0 to K115) . It should also be noted that nominally K⁺-free plates (K0) contain up to 20 µM traces of K⁺ due to contaminations of the chemicals used . Most of the strains carrying mutant derivatives of kdpA showed the same growth properties as TKW3205/pSM1 (wild-type kdpFABC) (data not shown) . TKW3205/pSM-kdpA:G233Y did not grow on K⁺ at concentrations less than 0.3 mM, and TKW 3205/pSD126 and TKW3205/pSR5 need at least 0.5 mM K⁺ for growth, whereas TKW3205/pSM-kdpA:G232A and TKW3205/pSM-kdpA:G232S required at least 5 mM K⁺ for growth (data not shown), suggesting a strong effect of the corresponding substitutions on the K⁺ affinity of the KdpFABC complex .

In vitro characterization of purified wild-type and mutant KdpFABC complexes. For induction of wild-type and mutant kdpFABC operons, strain TKW3205, after being transformed with one of the different plasmids, was grown in K0 medium or in medium with higher but limiting K⁺ concentrations, according to the influence of the particular amino acid substitution on the K⁺ affinity . The different KdpFABC complexes were purified as described previously (21) . To remove NH₄Cl, KdpFABC-containing fractions were dialyzed for 16 to 18 h at 4°C against a 200-fold volume of the buffer (50 mM Tris-HCl [pH 7.5], 20 mM MgCl₂, 10% glycerol, 0.2% Aminoxid WS 35), changed once . The cation-dependent ATPase activity, which is a common feature of P-ATPases (13, 15), was determined for the KdpFABC complexes as described previously (2) . The monovalent cations tested were K⁺, Rb⁺, Na⁺, Li⁺, Cs⁺, and NH₄⁺ . The factors by which saturating concentrations of the different cations stimulated or inhibited the basal ATPase activities and the cation concentrations, resulting in half-maximal stimulation or inhibition (K_0.5), were calculated and are summarized in Table 2 . For wild-type KdpFABC (Fig . 1A), K⁺ causes a stimulation factor of 4 and a K_0.5 of 39 µM was found . While Rb⁺ and Na⁺ were shown to slightly stimulate ATPase activity, Cs⁺ and Li⁺ inhibited ATPase activity ca . threefold . Interestingly, NH₄⁺ stimulated the ATPase activity by a factor of 2.2 with a K_0.5 of 3.9 mM, meaning that maximal activation was approximately half of that achieved with K⁺ but with a 100-fold lower affinity than that achieved with K⁺ . In contrast, complex Q116R (Fig . 1B), which shows a strongly reduced K⁺ affinity but the same maximal rate and cation selectivity as those of the wild-type enzyme (9, 18), was hardly stimulated by NH₄⁺ . (Purified KdpFABC complexes from kdpA mutant strains are named according to the amino acid substitution, e.g., complex G232D.) Complex G232D was first described by Buurman et al . (3) and more intensively analyzed by Schrader et al . (18) . The strongly reduced ability of this mutant enzyme to discriminate against Rb⁺ was confirmed (Fig . 1C) . A maximal rate similar to those observed using K⁺ and Rb⁺ was also observed in the presence of NH₄⁺ . Figure 1D shows the cation-dependent ATPase activity of complex G232A . No significant K⁺ stimulation of ATPase activity was observed at concentrations up to 25 mM, and no saturation was achieved, even at 200 mM . At higher concentrations, however, the stimulating effect of K⁺ overlaps with the inhibiting effect of high ionic strength, a general effect observed for all enzyme complexes tested (M . van der Laan, unpublished results); therefore, K_0.5 and maximum rate of metabolism values could not be determined properly . Maximal ATPase activity was 2,280 µmol · g^-1 · min^-1 (measured in the presence of 300 mM KCl), which represents a 1.8-fold stimulation . Calculated from this value, the K_0.5 would be 150 mM, which implies a 3,750-fold decrease of the K⁺ affinity at a minimum . In contrast, NH₄⁺ stimulated the ATPase activity of mutant G232A with the same affinity and rate as those of the wild-type complex (Table 2) . Rb⁺ was found to inhibit the ATPase activity of complex G232A ca . twofold . With complex G232S, comparable results were obtained (Fig . 1E) . Surprisingly, K⁺ inhibited ATPase activity of the purified G232S complex . However, when tested in inner-membrane vesicles, high K⁺ concentrations moderately stimulated ATPase activity (data not shown) . The wild-type enzyme, as well as all other mutant enzymes tested, did not show these discrepancies between the membrane-integrated and solubilized states . Substitutions of amino acids G233 and G234 (Table 2) only moderately affected the ion-binding properties of KdpA . The K⁺ affinity of complex G233Y (Fig . 1F) was reduced 10-fold, and the maximal activity was somewhat lower . For substitution G234D (Fig . 1H), a similar effect on affinity but an increased maximal activity were observed .

 
Concluding remarks. Based on sequence alignments with K⁺ channels and K⁺ symporters (7, 8, 11, 12) and on mutagenesis studies (3, 5), it has been suggested that the three conserved glycine residues in KdpA, G232, G233, and G234, form a selectivity filter-like structure similar to that found in the KcsA K⁺ channel (6) . In particular, G232 appeared to be of crucial importance for K⁺ affinity and cation selectivity . However, the only mutation in this region that has been characterized biochemically so far is G232D (3, 18) . We have systematically mutagenized this GGG motif and purified the altered KdpFABC complexes and characterized them by means of their cation-dependent ATPase activity . Using this biochemical approach, we have found that substitution G232S and even the rather conserved substitution G232A cause a dramatic decrease of K⁺ affinity and a complete loss of cation selectivity . This confirms and extends in vivo studies by Dorus et al . (5) and demonstrates that the growth phenotype of the corresponding mutants is indeed caused by an impaired K⁺ binding to KdpA . Our results stress that the K⁺ selectivity is mainly determined by G232, while a variety of amino acid substitutions is tolerated at positions 233 and 234 . It is therefore suggested that G232 is homologous to the highly conserved N-terminal selectivity filter glycine residue in the putative P-loops of K⁺ symporters, which, like KdpA, might be evolutionarily derived from K⁺ channels (8) . Furthermore, we show for the first time that NH₄⁺ also strongly stimulates the ATPase activity of the KdpFABC complex . Surprisingly, G232 substitutions only moderately affect NH₄⁺ affinities, indicating mechanistic differences between K⁺ and NH₄⁺ binding . It remains to be established whether KdpFABC can actually transport NH₄⁺, as has been suggested by Neijssel et al . (14) and Buurman et al . (4) on the basis of physiological studies .

This work was supported by the Deutsche Forschungsgemeinschaft (SFB431/P7) and the Fonds der Chemischen Industrie .

Present address: Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, 9751 NN Haren, The Netherlands .

Present address: Department of Pathochemistry, German Cancer Research Center, DKFZ, D-69120 Heidelberg, Germany .

1. Altendorf, K., and W . Epstein. 1996 . The Kdp-ATPase of Escherichia coli, p . 403-420 . In A . G . Lee (ed.), Biomembranes (ATPases), vol . 5 . JAI Press Inc., Greenwich, Conn.
2. Altendorf, K., M . Gaßel, W . Puppe, T . Möllenkamp, A . Zeeck, C . Boddien, K . Fendler, E . Bamberg, and S . Dröse. 1998 . Structure and function of the Kdp-ATPase of Escherichia coli . Acta Physiol . Scand . 163(Suppl.):137-146.
3. Buurman, E . T., K.-T . Kim, and W . Epstein. 1995 . Genetic evidence of two sequentially occupied K⁺ binding sites in the Kdp transport ATPase . J . Biol . Chem . 270:6678-6685.
4. Buurman, E . T., M . J . Teixera de Mattos, and O . M . Neijssel. 1991 . Futile cycling of ammonium ions via the high affinity potassium uptake system (Kdp) of Escherichia coli . Arch . Microbiol . 155:391-395.
5. Dorus, S., H . Mimaru, and W . Epstein. 2001 . Substrate-binding clusters of the K⁺-transporting Kdp ATPase of Escherichia coli investigated by amber suppression scanning mutagenesis . J . Biol . Chem . 276:9590-9598.
6. Doyle, D . A., J . M . Cabral, R . A . Pfuetzner, A . Kuo, J . M . Gulbis, S . L . Cohen, B . T . Chait, and R . MacKinnon. 1998 . The structure of the potassium channel: molecular basis of K⁺ conduction and cation selectivity . Science 280:69-77.
7. Durell, S . R., E . P . Bakker, and H . R . Guy. 2000 . Does the KdpA subunit from the high affinity K⁺-translocating P-type Kdp-ATPase have a structure similar to that of K⁺ channels? Biophys . J . 78:188-199.
8. Durell, S . R., Y . Hao, T . Nakamura, E . P . Bakker, and H . R . Guy. 1999 . Evolutionary relationship between K⁺ channels and symporters . Biophys . J . 77:775-788.
9. Fendler, K., S . Dröse, W . Epstein, E . Bamberg, and K . Altendorf. 1999 . The Kdp-ATPase of Escherichia coli mediates an ATP-dependent, K⁺-independent electrogenic partial reaction . Biochemistry 38:1850-1856.
10. Gaßel, M. 1999 . Charakterisierung, Reinigung und Rekonstitution des Kdp-Komplexes aus Escherichia coli unter besonderer Berücksichtigung der Untereinheiten KdpC und KdpF sowie Untersuchungen zur Identifikation der Plekomakrolidbindestelle von P- und V-ATPasen . Ph.D . thesis . University of Osnabrück, Osnabrück, Germany.
11. Jan, L . Y., and Y . N . Jan. 1994 . Potassium channels and their evolving gates . Nature 371:119-122.
12. Jan, L . Y., and Y . N . Jan. 1997 . Cloned potassium channels from eukaryotes and prokaryotes . Annu . Rev . Neurosci . 20:91-123.
13. Møller, J . V., B . Juul, and M . le Maire. 1996 . Structural organization, ion transport, and energy transduction of P-type ATPases . Biochim . Biophys . Acta 1286:1-51.
14. Neijssel, O . M., E . T . Buurman, and M . J . Teixera de Mattos. 1990 . The role of the futile cycles in the energetics of bacterial growth . Biochim . Biophys . Acta 1018:252-255.
15. Pedersen, P . L., and E . Carafoli. 1987 . Ion motive ATPases . I . Ubiquity, properties, and significance to cell function . Trends Biochem . Sci . 12:146-150.
16. Puppe, W. 1991 . Kalium-Transport bei Escherichia coli: Molekulargenetische und biochemische Untersuchungen zu funktionellen Domänen der Kdp-ATPase . Ph.D . thesis . University of Osnabrück, Osnabrück, Germany.
17. Puppe, W., A . Siebers, and K . Altendorf. 1992 . The phosphorylation site of the Kdp-ATPase of Escherichia coli: site-directed mutagenesis of the aspartic acid residues 300 and 307 of the KdpB subunit . Mol . Microbiol . 6:3511-3520.
18. Schrader, M., K . Fendler, E . Bamberg, M . Gassel, W . Epstein, K . Altendorf, and S . Dröse. 2000 . Replacement of glycine 232 by aspartic acid in the KdpA subunit broadens the ion specificity of the K⁺-translocating KdpFABC complex . Biophys . J . 79:802-813.
19. Siebers, A., and K . Altendorf. 1988 . The K⁺-translocating Kdp-ATPase from Escherichia coli . Purification, enzymatic properties and production of complex- and subunit-specific antisera . Eur . J . Biochem . 178:131-140.
20. Siebers, A., and K . Altendorf. 1989 . Characterization of the phosphorylated intermediate of the K⁺-translocating Kdp-ATPase from Escherichia coli . J . Biol . Chem . 264:5831-5838.
21. Siebers, A., R . Kollmann, G . Dirkes, and K . Altendorf. 1992 . Rapid, high-yield purification and characterization of the K⁺-translocating Kdp-ATPase from Escherichia coli . J . Biol . Chem . 267:12717-12721.
22. Sugiura, A., K . Nakashima, K . Tanaka, and T . Mizuno. 1992 . Clarification of the structural and functional features of the osmoregulated kdp operon of Escherichia coli . Mol . Microbiol . 6:1769-1776.
23. Yanisch-Perron, C., J . Vieira, and J . Messing. 1985 . Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors . Gene 33:103-119.

Free Online Full-text Article

What Is Bioreactor?, What Is Genome?, What Is Bioengineering?, What Is Antibiotic?, What Is Biofilter?, o, Bacterium, i, Bacteriology, c, Bacteria, i, Microorganism, n, Microbes, i, Microorganisms, a, Streptococcal, s, Pseudomonas, o, Cell suspensions, n, Multidrug resistant, o, Yeasts, e, Streptomycin, o, Microbial, s, Microorganisms, r, Microbial, r, Microorganism, e, Enterobacteriacea, r, Bacillus subtilis, a, Microorganisms, s, Streptococci, n, Schizosaccharomyces, a, Antimicrobial, r, Cell suspensions, c, Microbial, i, Microorganism, o, Gram positive




 

© 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com
 

Last modified: May 25, 2005