S-Glucuronidation of 7-mercapto-4-methylcoumarin by human UDP glycosyltransferases in genetically engineered fission yeast cells.

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.

Production of ibuprofen acyl glucosides by human UGT2B7.

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.

Production of human phase 1 and 2 metabolites by whole-cell biotransformation with recombinant microbes.

Cytochrome P450 enzymes (CYPs or P450s) are the most important enzymes involved in the phase I metabolism of drugs and poisons in humans, while UDP glycosyltransferases catalyze the majority of phase II reactions. In addition, a number of other enzymes or enzyme families contribute to the metabolism of xenobiotica, including alcohol dehydrogenase, aldehyde dehydrogenase, ester and amide hydrolases, epoxide hydrolase and flavine monooxygenases, as well as sulfotransferases, catechol-O-methyltransferase and N-acetyltransferase. A thorough understanding of their activity and of the properties of the metabolites they form is an essential prerequisite for the assessment of drug-caused side effects or toxicity. In this context of MIST, efficient production systems are needed to permit the large-scale production of human drug metabolites. As classical chemical synthesis cannot always provide these metabolites, biotechnological approaches have been developed that typically employ the recombinant expression of human drug-metabolizing enzymes. This review summarizes the current knowledge regarding whole-cell biotransformation processes that make use of such an approach.

Glucuronide production by whole-cell biotransformation using genetically engineered fission yeast Schizosaccharomyces pombe.

Drug metabolites generated by UDP glycosyltransferases (UGTs) are needed for drug development and toxicity studies, especially in the context of safety testing of metabolites during drug development. Because chemical metabolite synthesis can be arduous, various biological approaches have been developed; however, no whole-cell biotransformation with recombinant microbes that express human UGTs was yet achieved. In this study we expressed human UDP glucose-6-dehydrogenase together with several human or rat UGT isoforms in the fission yeast Schizosaccharomyces pombe and generated strains that catalyze the whole-cell glucuronidation of standard substrates. Moreover, we established two methods to obtain stable isotope-labeled glucuronide metabolites: the first uses a labeled aglycon, whereas the second uses (13)C(6)-glucose as a metabolic precursor of isotope-labeled UDP-glucuronic acid and yields a 6-fold labeled glucuronide. The system described here should lead to a significant facilitation in the production of both labeled and unlabeled drug glucuronides for industry and academia.