Uridine diphosphogalactofuranose (UDP-Galf ) is the precursor of the d-galactofuranose (Galf ) residues found in bacterial and parasitic cell walls, including those of many pathogens, such as Mycobacterium tuberculosis and Trypanosoma cruzi. UDP-Galf is made from UDP-galactopyranose (UDP-Galp) by the enzyme UDP-galactopyranose mutase (mutase). The mutase enzyme is essential for the viability of mycobacteria and is not found in humans, making it a viable therapeutic target. The mechanism by which mutase achieves the unprecedented ring contraction of a nonreducing sugar is unclear. We have solved the crystal structure of Escherichia coli mutase to 2.4 A resolution. The novel structure shows that the flavin nucleotide is located in a cleft lined with conserved residues. Site-directed mutagenesis studies indicate that this cleft contains the active site, with the sugar ring of the substrate UDP-galactose adjacent to the exposed isoalloxazine ring of FAD. Assay results establish that the enzyme is active only when flavin is reduced. We conclude that mutase most likely functions by transient reduction of substrate.

Carbamazepine (CBZ) is one of the most widely prescribed anticonvulsants despite a high incidence of idiosyncratic side effects. Metabolism of CBZ is complex, and of the more than 30 metabolites identified, one of the most abundant is CBZ Nglucuronide. To date the uridine diphosphate glucuronosyltransferase (UGT) isoform responsible for the N-glucuronidation of CBZ has not been identified. We have developed a sensitive liquid chromatography/mass spectrometry assay to quantify CBZ glucuronidation, and we report that CBZ is specifically glucuronidated by human UGT2B7. Kinetics of CBZ glucuronidation in human liver, kidney, and intestine microsomes were consistent with those of recombinant UGT2B7, which displayed a K m value of 214 M and V max value of 0.79 pmol/mg/min. In addition to revealing the isoform responsible for CBZ glucuronidation, this is the first example of primary amine glucuronidation by UGT2B7.Carbamazepine (5H-dibenzo[b,f]azepine-5-carboxamide) is one of the most widely prescribed anticonvulsants and is used to treat a variety of conditions from epilepsy to muscle spasm and trigeminal neuralgia. However its use is associated with a number of idiosyncratic adverse side effects, including skin rash, blood disorders, and hepatitis in onethird to one-half of patients (Ju and Uetrecht 1999). These adverse side effects have been associated with the formation of CBZ metabolites (Shear and Spielberg 1988;Riley et al., 1989); therefore, the study of all CBZ metabolites has important clinical implications.The metabolism of CBZ is complex and has been widely studied in human and in animal models (Madden et al., 1996;Maggs et al., 1997) with over 30 metabolites (Lertratanangkoon and Horning, 1982) identified. The major metabolites are the 10,11-epoxide and its hydrolytic trans-dihydrodiol product. Glucuronidation is also an important detoxification pathway because the CBZ N-glucuronide and glucuronides of the hydroxylated metabolites are significant urinary metabolites; however, to date no glucuronide metabolite has been implicated in the incidence of side effects. Epoxide hydrolase has been the focus of most attention as a potential source of toxic metabolites; however, the available data do not support a major role for this enzyme in causing side effects (Pirmohamed et al., 1992;Green et al., 1995b). One minor metabolite, 2-hydroxy-CBZ, formed through loss of the carboxamide, could be metabolized to an iminoquinone, which due to its potential chemical reactivity might be the metabolite responsible for the idiosyncratic reactions, although this has not been shown to date (Ju and Uetrecht, 1999). In addition CBZ is also a well known enzyme inducer up-regulating cytochrome P450 (Luo et al., 2002) and UGT activity/expression (Tanaka, 1999) Carbamazepine is metabolized to an N-glucuronide (Bauer et al., 1976); in addition, glucuronide metabolites have been demonstrated for all 13 of the hydroxylated metabolites of CBZ (Maggs et al., 1997). Formation of N-glucuronides has been principally studied in nonro...

No abstract

Farnesol is an isoprenoid found in many aromatic plants and is also produced in humans, where it acts on numerous nuclear receptors and has received considerable attention due to its apparent anticancer properties. Although farnesol has been studied for over 30 years, its metabolism has not been well characterized. Recently, farnesol was shown to be metabolized by cytochromes P450 in rabbit; however, neither farnesol hydroxylation nor glucuronidation in humans have been reported to date. In the present paper, we show for the first time that farnesol is metabolized to farnesyl glucuronide, hydroxyfarnesol and hydroxyfarnesyl glucuronide by human tissue microsomes, and we identify the specific human UGTs (uridine diphosphoglucuronosyltransferases) involved. Farnesol metabolism was examined by a sensitive LC (liquid chromatography)-MS/MS method. Results indicate that farnesol is a good substrate for glucuronidation in human liver, kidney and intestine microsomes (values in nmol/min per mg).Initial analysis using expressed human UGTs indicated that UGTs 1A1 and 2B7 were primarily responsible for glucuronidation in vitro, with significantly lower activity for all the other UGTs tested (UGTs 1A3, 1A4, 1A6, 1A9 and 2B4). Kinetic analysis and inhibition experiments indicate that, in liver microsomes, UGT1A1 is primarily responsible for farnesol glucuronidation; however, in intestine microsomes, UGT2B7 is probably the major isoform involved, with a very-low-micromolar K m . We also show the first direct evidence that farnesol can be metabolized to hydroxyfarnesol by human liver microsomes and that hydroxyfarnesol is metabolized further to hydroxyfarnesyl glucuronide. Thus glucuronidation may modulate the physiological and/or pharmacological properties of this potent signalling molecule.