Transport of C5 dicarboxylate compounds by Pseudomonas putida

Abstract

Induced glutarate and 2-oxoglutarate uptake and transport by P. putida were investigated in whole cells and membrane vesicles, respectively. Uptake of 2-oxoglutarate, but not glutarate, was against a concentration gradient to 1.7-fold greater than the initial extracellular concentration. Membrane vesicles transported 2-oxoglutarate and glutarate against gradients to intramembrane concentrations fivefold greater than the initial extravesicle concentrations. The rates of transport of both compounds were greatest in the presence of the artificial electron donor system phenazine methosulfate-ascorbate. Malate and D-lactate were the only naturally occurring compounds that served as electron donors. Uptake and transport were inhibited by KCN, NaN3, and 2,4-dinitrophenol. Kinetic parameters of transport were: glutarate, apparent K(m)-1.22 mM, V(max)-400 nmol/min per mg of membrane protein; 2-oxoglutarate, apparent K(m)-131 μM, V(max)-255 nmol/min per mg of membrane protein. Studies of competitive inhibition indicated a common system for transport of five C5 dicarboxylate compounds. The apparent K(m) and K(i) values with 2-oxoglutarate as a substrate placed the substrate affinity for transport in the order 2-oxoglutarate > glutarate > D-2-hydroxyglutarate and L-2-hydroxyglutarate > glutaconate.