Phenylalanine90 and phenylalanine93 are crucial amino acids within the estrogen binding site of the human UDP-glucuronosyltransferase 1A10

Abstract

Human UDP-glucuronosyltransferase 1A10 has been identified as the major isoform involved in the biotransformation of a wide range of phenolic substrates, including native estrogens and their oxidized metabolites. Our recent studies point to the F 90 -M 91 -V 92 -F 93 amino acid motif of UGT1A10, which was identified using photoaffinity labeling followed by LC-MS/MS analysis, as a key determinant of the binding of phenolic substrates. In this report, we have evaluated the role of F 90 , V 92 , and F 93 in the recognition of estrogens by UGT1A10 using site-directed mutagenesis. Kinetic studies using 5 mutants revealed that F 90 and F 93 are critical residues for the recognition of all estrogen substrates. The substitution of F 90 with alanine totally abolished the activity of this enzyme toward all the estrogens investigated. Overall, sequential removal for the aromatic (FtoL) and of the hydrophobic chain (FtoA and VtoA) from amino acids 90, 92, and 93 position effectively alters estrogen recognition. This demonstrates that individual features of the native and hydroxylated estrogens determine the specific binding properties of the compound within the binding site of the human UGT1A10 and the mutants. The resulting activities are completely abolished, unchanged, increased or decreased depending on the structures of both the mutant and the substrate. The novel identification of UGT1A10 as the major isoform involved in the glucuronidation of all estrogens and the discovery of the importance of the FMVF motif in the binding of steroids will help to elucidate the molecular mechanism of glucuronidation, resulting in the design of more effective estrogen-based therapies.

Identification of UDP-glucuronosyltransferase 1A10 in non-malignant and malignant human breast tissues

“…Information on the involvement of UGTs in the detoxification of estrogens is limited; however, recent findings suggest that glucuronidation by UGTs could directly promote the removal of native estrogens and their hydroxylated metabolites from the body [14,[20][21][22]. Recently, our laboratory has identified seven human recombinant UGTs, UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, and 2B7, as being involved in the biotransformation of native estrogens and their oxidative metabolites.…”

“…Recently, our laboratory has identified seven human recombinant UGTs, UGT1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, and 2B7, as being involved in the biotransformation of native estrogens and their oxidative metabolites. Of the UGTs identified, UGT1A10 has been established as the major UGT isoform that conjugates all the studied estrogens and UGT2B7 conjugated the genotoxic 4-OHE 1 with relatively high activity [20]. To date, UGT1A1, 1A3, 1A4, 1A8, 1A9, 2B4, 2B7, 2B15 and 2B28 have been characterized in breast tissues and/or breast cell lines [23][24][25][26][27][28][29].…”

Characterization of UDP-glucuronosyltransferase (UGT1A1) Promoter Polymorphisms and Gene Expression on Ethnicity, Stage of Disease, and Menopausal Status in Breast Cancer

“…Estrogen metabolism by UGTs is the major drug-metabolic pathway that results in the complete inactivation of estrogens and their hydroxylated metabolites [9–12]. Alterations in UGTs, resulting in decreased UGT expression and glucuronidation activity towards estrogens and their metabolites, has been suggested to play a potential role in breast cancer risk [14–16].…”

“…Alterations in UGTs, resulting in decreased UGT expression and glucuronidation activity towards estrogens and their metabolites, has been suggested to play a potential role in breast cancer risk [14–16]. Our previous published study demonstrated that several UGTs, including UGT1A1, are involved in the complete inactivation of both native estrogens and their oxidative metabolites, including the genotoxic 4-OH-E1 [12]. We also demonstrated for the first time that UGT1A10 was the major UGT involved in estrogen and estrogen metabolite conjugation [16].…”

“…UGTs have the catalytic capacity to inactivate estrogens and their oxidative metabolites to inactive metabolites [10–12]. UGTs also maintain the steady state concentrations of estrogens, and other sex steroids, in breast tissues [13].…”