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

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• 作者：Xicheng Wang ; Rafiquel I. Sarker ; Peter C. Maloney
• 刊名：Biochemistry
• 出版年：2006
• 出版时间：August 29, 2006
• 年：2006
• 卷：45
• 期：34
• 页码：10344 - 10350
• 全文大小：170K
• 年卷期：v.45,no.34(August 29, 2006)
• ISSN：1520-4995

文摘

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 bystudying OxlT function after cysteine residues were separately introduced at these positions. Withoutfurther treatment, both mutant proteins had a null phenotype when they were reconstituted intoproteoliposomes. By contrast, significant recovery of function occurred when proteoliposomes were treatedwith MTSEA (methanethiosulfonate ethylamine), a thiol-specific reagent that implants a positively chargedamino group. In each case, there was a 2-fold increase in the Michaelis constant (K_M) for oxalate self-exchange (from 80 to 160 M), along with a 5-fold (K355C) or 100-fold (R272C) reduction in V_maxcompared to that of the cysteine-less parental protein. Analysis by MALDI-TOF confirmed that MTSEAintroduced 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 sothat 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 insize via introduction of the SCH₂CH₂NH₃⁺ adduct, which generates a side chain that is ~1.85 Å longerthan that of lysine or arginine. These findings lead us to conclude that R272 and K355 are essentialcomponents of the OxlT substrate-binding site.