Abstract
The UDP-glucuronosyltransferase UGT2B7 is an important human UGT isoform that catalyzes the conjugation of many endogenous and exogenous compounds, among them opioids, resulting in the formation ofd-glucuronides. The binding site of the aglycone is located in the N-terminal half of the protein. Using NMR analysis, we demonstrate that the opioid binding site in UGT2B7 is within the 84 to 118 N-terminal amino acids. Three maltose binding protein-UGT2B7 fusion proteins, 2B7F3 and 2B7F4 incorporating the amino acids 24 to 118 and 24 to 96 of UGT2B7, respectively, and 2B7F5 incorporating amino acids 84 to 118 of UGT2B7 were expressed in Escherichia coliand purified by affinity chromatography. NMR analysis showed that morphine was bound to the fusion protein 2B7F3 with aKD value similar to theKD values obtained for the previously produced fusion proteins, which included amino acids 24 to 180. Morphine did not bind to 2B7F4, but it did bind to 2B7F5. Both NMR 1-D spectra and NOESY experiments indicated that the 2B7F5 protein was mediating magnetization transfer within the morphine. These results allowed us to predict and model a binding site within the amino acids 96 to 101 of UGT2B7. A mutant fusion protein 2B7F3 with the substitution D99A was produced, and the NMR spectroscopy analysis of the protein supported the model. A marked reduction of morphine binding was observed when the charged aspartate was substituted with alanine.
Footnotes
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This work was supported by National Institutes of Health grant GM26221 (to T.R.T.). Support from the College of Medicine, University of Iowa (to W.R.K.), is also acknowledged.
- Abbreviations:
- UGT
- UDP-glucuronosyltransferase
- MBP
- maltose binding protein
- NOESY
- nuclear Overhauser effect spectroscopy
- 2B7F1
- fusion protein of amino acids 24 to 180 from UGT2B7 and MBP
- 2B7F2
- fusion protein of amino acids 24 to 142 from UGT2B7 and MBP
- 2B7F3
- fusion protein of amino acids 24 to 118 from UGT2B7 and MBP
- 2B7F4
- fusion protein of amino acids 24 to 96 from UGT2B7 and MBP
- 2B7F5
- fusion protein of amino acids 84 to 118 from UGT2B7 and MBP
- Received June 14, 2002.
- Accepted October 15, 2002.
- The American Society for Pharmacology and Experimental Therapeutics
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