Analysis of substrate-binding elements in OxlT, the oxalate:formate antiporter of Oxalobacter formigenes.
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 (K(M)) for oxalate self-exchange (from 80 to 160 microM), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V(max) 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 SCH(2)CH(2)NH(3)(+) adduct, which generates a side chain that is approximately 1.85 A 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.
Wang
Xicheng
X
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Sarker
Rafiquel I
RI
Maloney
Peter C
PC
D017920:Antiporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport / Q000502:physiology; D005561:Formates / Q000737:chemistry* / Q000378:metabolism; D010070:Oxalates / Q000737:chemistry* / Q000378:metabolism; D020624:Oxalobacter formigenes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry* / Q000378:metabolism; D040681:Structural Homology, Protein
D017920:Antiporters; D001426:Bacterial Proteins; D005561:Formates; D010070:Oxalates; D011510:Proteolipids; C020485:proteoliposomes; C030544:formic acid