Substrate Specificity of the Escherichia coli 4-Aminobutyrate Carrier Encoded bygabP

摘要

Transport of 4-aminobutyrate into Eseherichia coli is catalyzed by gab permease(GabP). Although published studies show that GabP is relatively specific, recognizing the common a-amino acids with low affinity, recent work from this laboratory indicates that a number of synthetic compounds are high affinity transport inhibitors (50% inhibition at 5-100 uM). Here we present evidence that many of these structurally heterogeneous compounds not only inhibit transport but also function as alternative GabP substrates (i.e. a set of observations inconsistent with the idea that the core of the GabP transport channel exhibits rigid structural specificity for the native substrate, 4-aminobutyrate). The gab gene cluster is required for metabolism of 4-aminobutyrate in Escherichia coli. The cluster consists of a regulatory gene, gab C, two structural genes (gabD and gabT) encoding the metabolic enzymes, succinic semialdehyde dehydrogenase, and glutamate:succinic semialdehyde transaminase, and a third structural gene (gabP), encoding the 4-aminobutyrate transporter (gab permease or GabP). The GabP is a hydrophobic, 466-residue polypeptide (7) that is readily modeled as a transmembrane protein consisting of 12 transmembrane a-helical segments. The permease is active in whole cells as well as in rightside-out vesicles (3), and uptake of 4-aminobutyrate is stimulated by membrane potential and abolished by proton ionophores (7). Recently, we showed that a number of synthetic compounds are potent GabP inhibitors (8, 9). An unanswered question is whether any of these inhibitors might in fact be transported substrates of GabP. Here, we provide evidence consistent with the hypothesis that GabP transports at least nine different substrate analogs.