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Vol. 63, Issue 2, 283-288, February 2003
Department of Pharmacology (B.L.C., T.R.T.), NMR Facility (W.R.K.,
S.G.), College of Medicine, and ITS Research Technologies (B.M.K.),
University of Iowa, Iowa City, Iowa
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 of
D-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 coli
and purified by affinity chromatography. NMR analysis showed that
morphine was bound to the fusion protein 2B7F3 with a
KD value similar to the
KD 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.
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