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 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 K(D) value similar to the K(D) 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.
Subject(s)
Glucuronosyltransferase/metabolism , Morphine/pharmacology , Amino Acid Sequence , Animals , Aspartic Acid/genetics , Binding Sites , Carrier Proteins/biosynthesis , Carrier Proteins/genetics , Glucuronosyltransferase/chemistry , Glucuronosyltransferase/genetics , Humans , Magnetic Resonance Spectroscopy , Maltose-Binding Proteins , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Recombinant Fusion Proteins/biosynthesisABSTRACT
Mammalian UDP-glucuronosyltransferases are a family of isoenzymes that catalyze the reaction of endobiotics and xenobiotics with glucuronic acid resulting in the formation of hydrophilic glucuronides. This pathway is an important step in the metabolism and subsequent excretion of many compounds that would otherwise have toxic effects. This unit describes three methods for measuring UGT activity. Thin layer chromatography is a powerful screening method and may be used to analyze multiple substrates simultaneously. The Sep-Pak C18 cartridge extraction method has been developed to specifically separate opioid glucuronides from UDP-glucuronic acid. Finally, the ethyl acetate extraction method is used to separate the glucuronides of bilirubin, sterols, and vile acids from UDP-glucuronic acid. These methods may be applied to a microsomal fraction or to cultured cells transformed with cDNA for UGT.
