Amino Acid Residue Ile211 Is Essential for the Enzymatic Activity of Human Udp-Glucuronosyltransferase 1a10 (Ugt1a10)

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Conjugation of exogenous and endogenous compounds by uridine diphosphoglucuronosyltransferases (UGTs) is a pathway catalyzing the transfer of a glucuronic acid molecule from UDP glucuronic acid to lipophilic aglycones, which become more polar and more easily excretable in the bile or urine. UGTs are divided into two major families, UGT1 and UGT2. The isoform UGT1A10, along with UGT1A7 and UGT1A8, is expressed exclusively in extrahepatic tissues, notably in the gastrointestinal tract. Here, we report the isolation of a mutant clone of the human UGT1A10, at position 211 of the protein, where a threonine residue replaces an isoleucine residue (allele Thr211). Because the isoleucine is conserved among many UGT1A isoforms, we proceeded to the analysis of the activity of the wild-type UGT1A10 (T211I) and compared it with that of the variant enzyme (I211T*). In vitro assays with microsomal extracts from stably expressing human embryonic kidney 293 (HEK293) cells showed that the mutant enzyme lost all detectable activity toward major substrates, which demonstrate that the residue isoleucine at position 211 is essential for UGT1A10 activity. Mutant UGT1A10 (I211T*) also lost all detectable activity toward mycophenolic acid. Genomic DNA from 103 unrelated individuals was sequenced for this mutation, and two heterozygous genotypes were detected for this mutation (frequency: 2 per 100 individuals). Because UGT1A10 appears to be expressed in all gastrointestinal tissues and is active toward a wide range of substrates, lack of activity of this isoform may have an impact on individual glucuronidation efficiency.

Influence of N-Terminal Domain Histidine and Proline Residues on the Substrate Selectivities of Human UDP-Glucuronosyltransferase 1A1, 1A6, 1A9, 2B7, and 2B10

“…The latter observation and the loss of 4MU and 1NP glucuronidation activity associated with the UGT1A and UGT2B7 His3 Pro mutations are consistent with the hypothesis that the N-terminal domain histidine generally, but not invariably (Kubota et al, 2007;, acts as the catalytic base in the metabolism of compounds requiring proton abstraction for glucuronide formation. Taken together with previous reports (Dubois et al, 1999;Senay et al, 2002;Martineau et al, 2004;Xiong et al, 2006;Barre et al, 2007;Kubota et al, 2007;, the present study further demonstrates the pivotal roles of individual amino acids in determining the substrate selectivities of human UGT enzymes.…”

“…All UGT1A enzymes contain an identical carboxyl terminus of 246 residues but unique N-terminal domains (residues 1 to 285-289) , 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, 1992Ritter et al, , 1993Li 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;.…”

Analysis of R- and S-Hydroxywarfarin Glucuronidation Catalyzed by Human Liver Microsomes and Recombinant UDP-Glucuronosyltransferases

“…The broad tissue distribution and substrate specificity of UGT1A10 indicate that it may contribute to the extrahepatic detoxification of endogenous and xenobiotic compounds, especially orally administered drugs such as warfarin, and our past and current studies show that it is also involved in the glucuronidation of hydroxywarfarins Zielinska et al, 2008). Although there are many studies focused on the substrate specificity of UGT1A10, studies attempting to identify catalytically important amino acids and the characteristics of the binding site are less prevalent (Martineau et al, 2004;Basu et al, 2005;Xiong et al, 2006Xiong et al, , 2008Starlard-Davenport et al, 2007;Miller et al, 2008).…”

RESPONSE TO THE LETTER TO THE EDITOR BY DRS. N. PICARD AND P. MARQUET REGARDING A PUBLICATION: BASU ET AL., DRUG METABOLISM AND DISPOSITION (32:768–773, (2004)

“…3B). We also cite studies by other investigators who analyzed radiolabeled glucuronides following similar TLC resolution (Mackenzie, 2000;Martineau et al, 2004).…”

“…3B). We also cite studies by other investigators who analyzed radiolabeled glucuronides following similar TLC resolution (Mackenzie, 2000;Martineau et al, 2004).Why carry out differential pH measurements on UGT isozymes? Our aim was to conduct a biochemical study of the individual isozymes using optimal in vitro conditions.…”