Phenylalanine 90 and 93 Are Localized within the Phenol Binding Site of Human UDP-Glucuronosyltransferase 1A10 as Determined by Photoaffinity Labeling, Mass Spectrometry, and Site-Directed Mutagenesis

Abstract

4-Azido-2-hydroxybenzoic acid (4-AzHBA), a novel photoactive benzoic acid derivative, has been synthesized and used as a photoprobe to identify the phenol binding site of UDP-glucuronosyltransferases (UGTs). Analysis of recombinant His-tag UGTs from the 1A family for their ability to glucuronidate p-nitrophenol (pNP) and 4-methylumbelliferone (4-MU) revealed that UGT1A10 shows high activity toward phenols and phenol derivatives. Purified UGT1A10 was photolabeled with 4-AzHBA, digested with trypsin, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-mass spectrometry. A single modified peak corresponding to amino acid residues 89-98 (EFMVFHAQWK) of UGT1A10 was identified. The attachment site of the 4-AzHBA probe was localized to the quadruplet Phe(90)-Met(91)-Val(92)-Phe(93) using ESI LC-MS/MS. Sequence alignment revealed that the Phe(90) and Phe(93) are conserved in UGT1A7-10. Site-directed mutagenesis of these two amino acids was then followed by kinetic analysis of the mutants with two phenolic substrates, pNP and 4-MU, containing one and two planar rings, respectively. Using the combination of photoaffinity labeling, enzymatic digestion, MALDI-TOF and LC-MS mass spectrometry, and site-directed mutagenesis, we have determined for the first time that Phe(90) and Phe(93) are directly involved in the catalytic activity of UGT1A10 toward 4-MU and pNP.

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…Similarly, in the human sulfotransferase SULT1A1, substrate inhibition by dopamine was observed when Phe-247 was mutated to leucine and was interpreted to mean that this substitution created space for the binding of a second dopamine molecule (35). Finally, the UDPglucuronosyltransferase human UGT1A10 has an amino acid motif ( 90 FMVF 93 ) that is postulated to be involved in binding of its phenolic substrates through ring stacking with the phenylalanine residues (38). Three mutants, V92A, F93G, and F93A, all show substrate inhibition kinetics (36).…”

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

“…Yet, despite the large number of biotransformation reactions mediated by UGTs, relatively little is known about the critical amino acids involved in the direct interactions of these enzymes with naturally occurring substrates, drugs, or environmental pollutants. In our previous report, the two phenylalanine residues of the F 90 -M 91 -V 92 -F 93 motif located within the N-terminal domain of UGT1A10 were identified as being critical to the binding and glucuronidation of simple phenols [23]. This motif was identified using photoaffinity labeling followed by LCMS/ MS sequencing and site-directed mutagenesis.…”

“…This motif was identified using photoaffinity labeling followed by LCMS/ MS sequencing and site-directed mutagenesis. In those studies, two mutations of this motif, F90A and F93A, were generated and evaluated with two specific phenolic compounds, 4-nitrophenol and 4-methylumbelliferone [23]. The current study was undertaken to further clarify the role of the previously identified FMVF motif of UGT1A10, and analysis of the two previously generated mutants as well as 3 new mutants, F90L, F93L, and V92A, revealed that the two amino acids, F 90 and F 93 , are also critical for the glucuronidation of native estrogens and their hydroxylated derivatives, while V 92 only decreases the activity of this enzyme toward E 3 .…”

“…Microsomal protein (5 μg) from baculovirus-infected Sf9 insect cells, which were engineered to express individual UGT enzymes, was used for all the 1A family proteins. This same set of enzymes has been used in previous studies from our lab and have been show to have a high catalytic capacity for a large variety of compounds, allowing for kinetic analyses be carried out under the optimal conditions for each isoform [23,25,27,28]. UGT2B7 was over-expressed in HK293 cells.…”

“…Moreover, since there is no information available on the structure of the estrogen binding site of this enzyme, or of other enzymes and/or nuclear receptors that bind these compounds, identification of the estrogen binding site of UGT1A10 was undertaken. Using photoaffinity labeling, specific phenol probes, and LC-MS/MS sequencing, the F 90 -M 91 -V 92 -F 93 amino acid motif was identified as the phenol binding site of UGT1A10 [23]. Several mutations of this motif were carried out by site-directed mutagenesis to confirm the importance of this site in phenol binding, and in the present studies, these same mutated enzymes were investigated for their ability to bind 7 different estrogens.…”