PcaK, a high-affinity permease for the aromatic compounds 4-hydroxybenzoate and protocatechuate from Pseudomonas putida.
PcaK is a transporter and chemoreceptor protein from Pseudomonas putida that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation. When expressed in Escherichia coli, PcaK was localized to the membrane and catalyzed the accumulation of two aromatic substrates, 4-hydroxybenzoate and protocatechuate, against a concentration gradient. Benzoate inhibited 4-hydroxybenzoate uptake but was not a substrate for PcaK-catalyzed transport. A P. putida pcaK mutant was defective in its ability to accumulate micromolar amounts of 4-hydroxybenzoate and protocatechuate. The mutant was also impaired in growth on millimolar concentrations of these aromatic acids. In contrast, the pcaK mutant grew at wild-type rates on benzoate. The Vmax for uptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the Km was 6 microM. PcaK-mediated transport is energized by the proton motive force. These results show that although aromatic acids in the undissociated (uncharged) form can diffuse across bacterial membranes, high-specificity active transport systems probably also contribute to the ability of bacteria to grow on the micromolar concentrations of these compounds that are typically present in soil. A variety of aromatic molecules, including naturally occurring lignin derivatives and xenobiotics, are metabolized by bacteria and may be substrates for transport proteins. The characterization of PcaK provides a foundation for understanding active transport as a critical step in the metabolism of aromatic carbon sources.
Nichols
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NN
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
Harwood
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CS
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001565:Benzoates / Q000378:metabolism; D019817:Benzoic Acid; D001693:Biological Transport, Active; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D062385:Hydroxybenzoates / Q000378:metabolism*; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D010226:Parabens / Q000378:metabolism*; D018892:Proton-Motive Force; D016958:Pseudomonas putida / Q000201:enzymology / Q000254:growth & development / Q000378:metabolism*
D001426:Bacterial Proteins; D001565:Benzoates; D002352:Carrier Proteins; D062385:Hydroxybenzoates; D026901:Membrane Transport Proteins; D010226:Parabens; C090106:PcaK protein, Pseudomonas; C009091:protocatechuic acid; D019817:Benzoic Acid; C038193:4-hydroxybenzoic acid