Molecular and biochemical studies on OxlT structure and function

The OxlT is a member of Major Facilitator Superfamily found in Oxalobacter formigenes. After the oxlT gene was cloned and expressed in Escherichia coli, the availability of high amounts of OxlT protein has expedited the study of OxlT structure and function.; A homology model for OxIT was generated based on the X-ray structure of the glycerol 3-phosphate transporter. We examined OxlT helix proximity by disulfide trapping to optimize the process so that the final model is consistent with all earlier biochemical data, and suggests that Arg272 serves as a positive center for substrate binding. We verified that Arg 272 is essential for function by evaluating the transport activity of mutants at 272 position.; In an extended study on OxlT substrate-binding elements, we confirmed that both Arg272 and Lys355 are critical to substrate transport. The cysteine-substitution proteins at these two positions showed a null phenotype, but each showed significant recovery of function when these proteins were modified by a thiol-specific reagent that implants a positively charged amino group at 272 and 355 positions. MALDI-TOF demonstrated that such treatment introduced the desired modification to the mutant proteins, and immunoblot analysis of the stability of solubilized OxlT indicated that such modification affected the entire OxlT population. In other work, we discovered that the modification also altered the substrate specificity.; To further examine the homology model, we constructed a single cysteine panel of TM5. We identified that eleven mutants responded to the thiol-specific probes. In additional tests, we observed three biochemical phenotypes. Group 1 displayed substrate protection against probe inhibition. Group 2 had unexpected responses in which modification led to increased activity if weak acids/bases or neutral probes were used, while modification by probes introducing a fixed charge led to decreased function. In both cases, the presence of substrate prevented the response to probes. Group 3 showed a probe sensitivity that was increased by the presence of substrate. The findings of substrate-protectable probe modification (Group 1 and Group 2) suggest that TM5 lies on the permeation pathway. In all, our biochemical data of TM5 is consistent with the OxlT homology model.