Influence of N-Terminal Domain Histidine and Proline Residues on the Substrate Selectivities of Human UDP-Glucuronosyltransferase 1 A 1 , 1 A 6 , 1 A 9 , 2 B 7 , and 2 B 10

An N-terminal domain histidine [corresponding to position 39 of UDP-glucuronosyltransferase (UGT) 1A1] is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobioticmetabolizing enzymes, UGT1A4 and UGT2B10 lack the ability to glucuronidate 4-methylumbelliferone (4MU) and 1-naphthol (1NP), both planar phenols, and naproxen (a carboxylic acid). However, only UGT1A4 glucuronidates the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP). In this study, we sought to elucidate the influence of specific N-terminal histidine and proline residues on UGT enzyme substrate selectivity. The conserved N-terminal domain histidine of UGT1A1, UGT1A6, UGT1A9, and UGT2B7 was mutated to proline and leucine 34 of UGT2B10 was substituted with histidine, and the capacity of the wild-type and mutant proteins to glucuronidate 4MU, 1NP, LTG, TFP, and naproxen was characterized. Whereas UGT1A1(H39P), UGT1A6(H38P), and UGT1A9(H37P) lacked the ability to metabolize 4MU, 1NP, and naproxen, all glucuronidated LTG. Km values for UGT1A1(H39P) and UGT1A9(H37P) were 774 and 3812 M, respectively, compared with 1579 M for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a Vmax/Km value comparable to that of UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective Km values of 260 and 118 M. UGT2B7(H35P) lacked activity toward all substrates. The data confirm a pivotal role for an N-terminal domain proline in the glucuronidation of the tertiary amines LTG and TFP by UGT1A subfamily proteins, whereas glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes. UDP-glucuronosyltransferase (UGT) enzymes catalyze the covalent linkage of glucuronic acid, donated by the cofactor UDP-glucuronic acid (UDPGA), to a typically hydrophobic substrate bearing a suitable functional group according to a second-order nucleophilic substitution mechanism. Hydroxyl (aliphatic and aromatic), carboxylic acid, and amine (primary, secondary, and tertiary) functional groups most commonly serve as the nucleophilic “acceptor” for glucuronic acid (Miners and Mackenzie, 1991; Radominska-Pandya et al., 1999). Because a myriad of compounds fulfill these minimal requirements for metabolism by UGT, glucuronidation provides an elimination and detoxification pathway for many drugs, nondrug xenobiotics, and endogenous compounds. Nineteen functional human UGT proteins have been identified to date, and these have been classified in two families (1 and 2) and three subfamilies (UGT1A, UGT2A, and UGT2B) based on sequence identity (Mackenzie et al., 2005). The individual UGT enzymes exhibit distinct, but overlapping, substrate and inhibitor selectivities (Radominska-Pandya et al., 1999; Tukey and Strassburg, 2000; Miners et al., 2004, 2006; Kiang et al., 2005; Sorich et al., 2006). The UDPGA binding site is highly conserved and located in the C-terminal domain of UGTs (Miley et al., 2007; Patana et al., 2007). Although an X-ray crystal structure for an entire UGT protein is lacking, multiple lines of evidence implicate the N-terminal domain in substrate (aglycone) binding and selectivity. All UGT1A enzymes contain an identical carboxyl terminus of 246 residues but unique N-terminal domains (residues 1 to 285–289) (Mackenzie et al., 2005), indicating that the latter region must determine substrate selectivity. Studies with chimeric UGT2B proteins have similarly associated the N-terminal domain with the substrate selectivities of several human, rabbit, and rat UGT2B enzymes (Mackenzie, 1990; Ritter et al., 1992, 1993; Li et al., 1997; Lewis et al., 2007), whereas site-directed mutagenesis has identified specific N-terminal domain residues involved in the binding of substrates of human UGT1A3, 1A4, 1A6, 1A10, 2B4, 2B15, and 2B17 (Dubois et al., 1999; Senay et al., 2002; Martineau et al., 2004; Xiong et al., 2006; Barre et al., 2007; Kubota et al., 2007; Li et al., 2007). Particularly striking are differences in the abilities of UGT1A and This work was supported by the National Health and Medical Research Coun-