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Drug Metabolism and Disposition Fast Forward
First published on June 1, 2009; DOI: 10.1124/dmd.109.028225

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Received for publication April 24, 2009.
Revised May 27, 2009.
Accepted for publication May 28, 2009.

Influence of N-terminal domain histidine and proline residues on the substrate selectivties of human UDP-glucuronosyltransferase (UGT) 1A1, 1A6, 1A9, 2B7 and 2B10

Oranun Kerdpin 1, Peter I Mackenzie 2, Kushari Bowalgaha 2, Moshe Finel 3, John O Miners 2*

1 Naresuan University and Flinders University 2 Flinders University 3 University of Helsinki

* Address correspondence to: E-mail: john.miners{at}flinders.edu.au

Abstract

An N-terminal domain histidine (corresponding to position 39 of UGT1A1) is conserved in all UGT1A and UGT2B subfamily proteins except UGT1A4 (Pro-40) and UGT2B10 (Leu-34). Unlike most UGT1A and UGT2B xenobiotic metabolizing 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). This study 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-40 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 µM and 3812 µM, respectively, compared to 1579 µM for UGT1A4. UGT1A1(H39P) also glucuronidated TFP with a Vmax/Km value comparable to UGT1A4. In contrast to the wild-type enzyme, UGT2B10(L34H) glucuronidated 4MU and 1NP with respective Km values of 260 µM and 118 µM. UGT2B7(H35P) lacked activity towards 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 while glucuronidation reactions involving proton abstraction generally, although not invariably, require a histidine at the equivalent position in both UGT1A and UGT2B enzymes.


Key words: glucuronidation, site-directed mutagenesis, structure-activity relationships, UDP glucuronyltransferases




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