Critical Roles of Residues 36 and 40 in the Phenol and Tertiary Amine Aglycone Substrate Selectivities of UDP-Glucuronosyltransferases 1A3 and 1A4 Takahiro Kubota, 1 Benjamin C. Lewis, David J. Elliot, Peter I. Mackenzie, and John O. Miners Department of Clinical Pharmacology, Flinders University and Flinders Medical Centre, Adelaide, Australia Received May 8, 2007; accepted July 16, 2007 ABSTRACT Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the pla- nar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4- MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glu- curonides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged be- tween UGT1A3 and UGT1A4 to generate UGT1A3-4 (45–535) , UGT1A3-4 (45–154) -3, UGT1A4-3 (45–535) , and UGT1A4-3 (45–154) -4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, UGT1A4-3 (45–535) and UGT1A4-3 (45–154) -4 [but not UGT1A3- 4 (45–535) and UGT1A3-4 (45–154) -3] retained the capacity to glu- curonidate LTG and TFP. Likewise, UGT1A3-4 (45–535) and UGT1A3-4 (45–154) -3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3 (45–535) and UGT1A4-3 (45–154) -4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. “Reciprocal” mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 re- duced in vitro clearances for LTG and TFP glucuronidation by 90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronida- tion by 90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glu- curonidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover. Glucuronidation involves the covalent linkage of glucu- ronic acid, derived from the cofactor UDP-glucuronic acid (UDPGA), to a substrate bearing a nucleophilic functional group or atom, most commonly an alcohol (aliphatic or phe- nolic), amine, or carboxylic acid. Given the widespread occur- rence of these functional groups in synthetic and “biological” chemicals, glucuronidation is not surprisingly an essential clearance and detoxification mechanism for a myriad of com- pounds that include drugs, environmental chemicals, and endogenous compounds particularly bilirubin, fatty acids, and hydroxysteroids (Miners and Mackenzie 1991; Radomin- ska-Pandya et al., 1999). The glucuronidation reaction is catalyzed by the UDP-glucuronosyltransferase (UGT) super- family of enzymes. Almost 30 UGT genes have been identi- fied in humans, and these have been classified in three sub- families: 1A, 2A, and 2B (Mackenzie et al., 2005). UGT1A and UGT2B enzymes, which are the largest subfamilies, are of greatest importance in the metabolism of xenobiotics and endogenous compounds in humans. It is well established that the individual UGT1A and UGT2B enzymes exhibit distinct but overlapping aglycone substrate selectivities and differ in terms of regulation of expression (Tukey and Strassburg 2000; Radominska-Pandya et al., 1999; Miners et al., 2004; Kiang et al., 2005). However, the structural basis of UGT enzyme substrate selectivity has yet to be fully elucidated. UGT1A enzymes comprise a unique amino terminus do- main of 285 to 289 residues but an identical carboxyl termi- nus comprising 246 residues that arise from the splicing of This work was supported by a grant from the National Health and Medical Research Council of Australia. P.I.M. is a National Health and Medical Re- search Council of Australia Senior Principal Research Fellow. T.K. was sup- ported by an Overseas Study Scholarship from the Japanese Research Foun- dation. 1 Current affiliation: Department of Drug Metabolism and Biopharmaceu- tics, Faculty of Pharmacy, Chiba Institute of Science, Chiba, Japan. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.107.037952. ABBREVIATIONS: UDPGA, UDP-glucuronic acid; UGT, UDP-glucuronosyltransferase; LTG, lamotrigine; TFP, trifluoperazine; 4-MU, 4-methyl- umbelliferone; 1-NP, 1-naphthol; HEK, human embryonic kidney; HPLC, high-performance liquid chromatography. 0026-895X/07/7204-1054 –1062$20.00 MOLECULAR PHARMACOLOGY Vol. 72, No. 4 Copyright © 2007 The American Society for Pharmacology and Experimental Therapeutics 37952/3257426 Mol Pharmacol 72:1054–1062, 2007 Printed in U.S.A. 1054 at ASPET Journals on January 8, 2017 molpharm.aspetjournals.org Downloaded from