Analysis of Substrate-Binding Elements in OxlT, the Oxalate:Formate Antiporter of Oxalobacter formigenes

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Abstract
An OxlT homology model suggests R272 and K355 in transmembrane helices 8 and 11, respectively, are critical to OxlT-mediated transport. We offer positive evidence supporting this idea by studying OxlT function after cysteine residues were separately introduced at these positions. Without further treatment, both mutant proteins had a null phenotype when they were reconstituted into proteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treated with MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively charged amino group. In each case, there was a 2-fold increase in the Michaelis constant (KM) for oxalate self-exchange (from 80 to 160 μM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in Vmax compared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEA introduced the desired modification. We also examined substrate selectivity for the treated derivatives. While oxalate remained the preferred substrate, there was a shift in preference among other substrates so that the normal rank order (oxalate > malonate > formate) was altered to favor smaller substrates (oxalate > formate > malonate). This shift is consistent with the idea that the substrate-binding site is reduced in size via introduction of the SCH2CH2NH3+ adduct, which generates a side chain that is ∼1.85 Å longer than that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essential components of the OxlT substrate-binding site.