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1.
Biol Chem ; 392(12): 1089-95, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22050224

RESUMO

Human UDP glycosyltransferases (UGTs) play an important role in xenobiotic detoxification. They increase the solubility of their substrates by adding a sugar moiety (such as glucuronic acid) to different functional entities (such as hydroxyl groups). The aim of this study was to investigate how glucuronidation of a standard substrate is affected by a change of the hetero-atom at the conjugation site. For this purpose, we compared the in vitro glucuronidation rates of 4-methylumbelliferone and 7-mercapto-4-methylcoumarin, respectively. Human liver microsomes catalyzed the S-glucuronidation of 7-mercapto-4--methylcoumarin almost as efficient as the O-glucuronidation of 4-methylumbelliferone. When testing isoenzyme specificity by whole cell biotransformation with fission yeast strains that recombinantly express all 19 human members of the UGT1 and UGT2 families, it was found that 13 isoenzymes were able to glucuronidate 7-mercapto-4-methylcoumarin, with five of them being specific for this substrate and the other eight also converting 4-methylumbelliferone under these conditions. The remaining six UGTs did not accept either substrate. Out of the eight isoenzymes that glucuronidated both substrates, four catalyzed both reactions approximately to the same extent, while three displayed higher conversion rates towards 4-methylumbelliferone and one preferred 7-mercapto-4-methylcoumarin. These data suggest that 7-mercapto-4-methylcoumarin is a convenient new standard substrate for monitoring S-glucuronidation.


Assuntos
Cumarínicos/metabolismo , Engenharia Genética , Glucuronídeos/metabolismo , Glicosiltransferases/metabolismo , Schizosaccharomyces/metabolismo , Cumarínicos/química , Glicosiltransferases/genética , Humanos , Microssomos Hepáticos/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética
2.
Drug Metab Dispos ; 39(12): 2174-81, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21862693

RESUMO

UDP-glycosyltransferases (UGTs) are an important group of enzymes that participate in phase II metabolism of xenobiotics and use the cofactor UDP-glucuronic acid for the production of glucuronides. When acting on molecules bearing a carboxylic acid they can form acyl glucuronides, a group of metabolites that has gained significant interest in recent years because of concerns about their potential role in drug toxicity. In contrast, reports about the production of drug acyl glucosides (which might also display high reactivity) have been scarce. In this study, we discovered the formation of acyl glycoside metabolites of R- and S-ibuprofen (Ibu) by human liver microsomes supplied with the cofactor UDP-glucose. Subsequently, human UGT2B7*1 and UGT2B7*2 recombinantly expressed in fission yeast Schizosaccharomyces pombe could be shown to catalyze these reactions. Moreover, we could enhance the glucoside production rate in fission yeast by overexpressing the fission yeast gene SPCC1322.04, a potential UDP-glucose pyrophosphorylase (UGPase), but not by overexpression of SPCC794.10, and therefore suggest to name this gene fyu1 for fission yeast UGPase1. It was interesting to note that pronounced differences between the two polymorphic UGT2B7 variants were observed with respect to acyl glucoside production. Finally, using the metabolic precursor [(13)C(6)]glucose, we demonstrated the production of stable isotope-labeled reference standards of Ibu acyl glucoside and Ibu acyl glucuronide by whole-cell biotransformation in fission yeast.


Assuntos
Glucosídeos/biossíntese , Glucuronosiltransferase/metabolismo , Ibuprofeno/metabolismo , Sequência de Bases , Biomassa , Cromatografia Líquida , Primers do DNA , Humanos , Espectrometria de Massas , Proteínas Recombinantes/genética , Schizosaccharomyces/genética
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