Nichols N N, Harwood C S
Department of Microbiology, University of Iowa, Iowa City 52242, USA.
J Bacteriol. 1997 Aug;179(16):5056-61. doi: 10.1128/jb.179.16.5056-5061.1997.
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.
PcaK是恶臭假单胞菌中的一种转运蛋白和化学感受器蛋白，它作为β-酮己二酸途径调节子的一部分被编码，用于芳香酸的降解。当在大肠杆菌中表达时，PcaK定位于细胞膜，并催化两种芳香底物4-羟基苯甲酸和原儿茶酸逆浓度梯度积累。苯甲酸抑制4-羟基苯甲酸的摄取，但不是PcaK催化转运的底物。恶臭假单胞菌的pcaK突变体在积累微摩尔量的4-羟基苯甲酸和原儿茶酸的能力上存在缺陷。该突变体在以毫摩尔浓度的这些芳香酸为底物的生长过程中也受到损害。相比之下，pcaK突变体在苯甲酸上以野生型速率生长。4-羟基苯甲酸摄取的Vmax至少为25 nmol/分钟/毫克蛋白质，Km为6 μM。PcaK介导的转运由质子动力提供能量。这些结果表明，尽管未离解（不带电荷）形式的芳香酸可以扩散穿过细菌细胞膜，但高特异性的主动转运系统可能也有助于细菌在土壤中通常存在的这些化合物的微摩尔浓度下生长的能力。包括天然存在的木质素衍生物和外源化合物在内的多种芳香分子可被细菌代谢，并且可能是转运蛋白的底物。PcaK的特性为理解主动转运作为芳香碳源代谢中的关键步骤提供了基础。
