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.
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.
Xiong
Yan
Y
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.
Bernardi
Dan
D
Bratton
Stacie
S
Ward
Michael D
MD
Battaglia
Eric
E
Finel
Moshe
M
Drake
Richard R
RR
Radominska-Pandya
Anna
A
D000595:Amino Acid Sequence; D001386:Azides / Q000737:chemistry; D001665:Binding Sites; D002853:Chromatography, Liquid; D014453:Glucuronosyltransferase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics*; D006801:Humans; D006923:Hymecromone / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009596:Nitrophenols / Q000378:metabolism; D019800:Phenol / Q000378:metabolism; D010649:Phenylalanine / Q000737:chemistry*; D019920:Photoaffinity Labels; D011994:Recombinant Proteins / Q000378:metabolism; D012459:Salicylates / Q000737:chemistry; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D001386:Azides; D009596:Nitrophenols; D019920:Photoaffinity Labels; D011994:Recombinant Proteins; D012459:Salicylates; D019800:Phenol; D006923:Hymecromone; D010649:Phenylalanine; C063873:4-azidosalicylic acid; C476725:bilirubin uridine-diphosphoglucuronosyl transferase 1A10; D014453:Glucuronosyltransferase; C024836:4-nitrophenol