A common polymorphic variant of UGT1A5 displays increased activity due to optimized cofactor binding

Abstract

Uridine diphosphate-glucuronosyltransferases (UGTs) are the most important phase II enzymes in human drug metabolism. Using permeabilized recombinant fission yeast cells (enzyme bags), we demonstrate that UGT1A5 can catalyze an N-glucuronidation reaction. We characterized two new polymorphic UGT1A5 variants: a common ninefold mutant (UGT1A5*8) with double-fold activity and a much rarer sixfold mutant (UGT1A5*9), which has the same activity as the wild-type. Molecular modeling studies indicate that the minor effects of all mutations, except for Gly259Arg, are due to their distance to the substrate binding site. Extensive molecular dynamics simulations revealed that the Gly259Arg mutation stabilizes helix Q through a newly formed hydrogen bonding network, which places the cofactor in a much more favorable geometry in UGT1A5*8 as compared to the wild-type.

Functional Expression of All Human Sulfotransferases in Fission Yeast, Assay Development, and Structural Models for Isoforms SULT4A1 and SULT6B1

“…Recently, we reported the successful use of permeabilized fission yeast cells (enzyme bags) for biotransformations catalyzed by human CYPs [43] or UGTs [17]. Such an assay design has the advantages of higher sensitivity and shorter reaction times, as substrates, cofactors, and products are not hampered by various biological membranes but can freely move between the assay medium and the inside of the cells.…”

“…This was essentially done as described in [17] with slight modifications. Briefly, fission yeast strains were grown in 10 mL liquid culture of EMM with supplements as needed at 30 • C and 230 rpm for 24 h. For each assay, 5 × 10 7 cells were transferred to 1.5 mL Eppendorf tubes, pelleted, and incubated in 1 mL of 0.3% Triton-X100 in Tris-KCl buffer (200 mM KCl, 100 mM Tris-Cl pH 7.8) at room temperature for 60 minutes at 150 rpm to allow permeabilization.…”

“…LC-MS chromatograms are shown for SULT1A3, SULT1E1, and SULT1B1, respectively (Figure 1). An additional assay format was developed which employs permeabilized fission yeast cells (enzyme bags) using a protocol similar to those previously reported for CYPs [43] and UGTs [17], but with the addition of the cofactor PAPS instead of NADPH or GSA. In these experiments SULT4A1 catalyzed the sulfation of 1-naphthol ( Figure 2), but not of 4-nitrophenol, 7-hydroxycoumarin, or DHEA.…”

“…The latter was reported to metabolize thyroxine and bithionol [12]. Previously, we successfully used fission yeast Schizosaccharomyces pombe for the functional expression of orphan cytochrome P450 (CYP) enzymes, such as CYP2A7, CYP4Z1, CYP4A22, and CYP20A1 [13][14][15][16], and UGTs such as UGT1A5 [17]. Homology models of these understudied enzymes guided by substrate activity data helped to increase knowledge on their functionality.…”

Drug and Chemical Glucosidation by Control Supersomes and Membranes from Spodoptera frugiperda (Sf) 9 Cells: Implications for the Apparent Glucuronidation of Xenobiotics by UDP-glucuronosyltransferase 1A5

“…100 pmol/16 h×mg, equivalent to 0.1 pmol/min×mg), and a nonspecific radiometric thin layer chromatographic method was used for product quantification (Ciotti et al, 1999). More recently, Yang et al (2018) described the expression of active UGT1A5 and two polymorphic variants (UGT1A5*8 and UGT1A5*9) in fission yeast (Schizosaccharomyces pombe) cells. The activities of Triton X-100 permeabilized cells expressing the UGT1A5 enzymes were investigated using the UGT-Glo assay (Promega, Madison, WI), which measures the depletion of proluciferin substrates (UGT-Glo substrates A and B) rather than metabolite formation.…”