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 putid a that is encoded as part of the beta-ketoadipate pathway regulon for aromatic acid degradation, When expressed in Escherichia coli, PcaK wa s localized to the membrane and catalyzed the accumulation of two arom atic substrates, 4-hydroxybenzoate and protocatechuate, against a conc entration gradient, Benzoate inhibited 4-hydroxybenzoate uptake but wa s not a substrate for PcaK-catalyzed transport, A P. putida pcaK mutan t was defective in its ability to accumulate micromolar amounts of 4-h ydroxybenzoate and protocatechuate, The mutant was also impaired in gr owth on millimolar concentrations of these aromatic acids, In contrast , the pcaK mutant grew at wild-type rates on benzoate, The V-max for u ptake of 4-hydroxybenzoate was at least 25 nmol/min/mg of protein, and the K-m was 6 mu M. PcaK-mediated transport is energized by the proto n motive force, These results short that although aromatic acids in th e undissociated (uncharged) form can diffuse across bacterial membrane s, 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 arom atic molecules, including naturally occurring lignin derivatives and x enobiotics, are metabolized by bacteria; and may be substrates for tra nsport proteins, The characterization of PcaK provides a foundation fo r understanding active transport as a critical step in the metabolism of aromatic carbon sources.