1180-25-2Relevant articles and documents
Glucuronide production by whole-cell biotransformation using genetically engineered fission yeast Schizosaccharomyces pombe
Dragan, Calin-Aurel,Buchheit, Daniela,Bischoff, Daniel,Ebner, Thomas,Bureik, Matthias
, p. 509 - 515 (2010)
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 13C6-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.
Experimental and kinetic studies of the Escherichia coli glucuronylsynthase: An engineered enzyme for the synthesis of glucuronide conjugates
Wilkinson, Shane M.,Watson, Morgan A.,Willis, Anthony C.,McLeod, Malcolm D.
supporting information; experimental part, p. 1992 - 2000 (2011/06/19)
The detection and study of glucuronide metabolites is essential in many fields including pharmaceutical development, sports drug testing, and the detection of agricultural residues. Therefore, the development of improved methods for the synthesis of glucuronide conjugates is an important aim. The glycosynthase derived from E. coli β-glucuronidase provides an efficient, scalable, single-step synthesis of β-glucuronides under mild conditions. In this article we report on experimental and kinetic studies of the E. coli glucuronylsynthase, including the influence of acceptor substrate, pH, temperature, cosolvents, and detergents, leading to optimized conditions for glucuronide synthesis. Enzyme kinetics also reveals that both substrate and product inhibition may occur in glucuronylsynthase reactions but that these effects can be ameliorated through the judicious choice of acceptor and donor substrate concentrations. An investigation of temporary polar substituents was conducted leading to improved aqueous solubility of hydrophobic steroidal acceptors. In this way the synthesis of the steroidal metabolite dehydroepiandrosterone 3-β-d-glucuronide was achieved in three steps and 86% overall yield from dehydroepiandrosterone.
