Human UDP-glucuronosyltransferase expression in insect cells: ratio of active to inactive recombinant proteins and the effects of a C-terminal his-tag on glucuronidation kinetics.

Many laboratories use recombinant UDP-glucuronosyltransferases (UGTs), expressed in baculovirus-infected insect cells, for drug glucuronidation studies. We have infected Sf9 insect cells with increasing amounts of recombinant baculovirus, encoding either UGT1A9 or UGT2B7, and measured both glucuronidation activity and immunodetectable UGT in the resulting cell homogenates. The correlation between glucuronidation rates and degree of infection followed different trends, depending on whether activity was the actual activity measured or was corrected for UGT expression level. Above a certain low level of infection, further increases in infection ratios led to a large decline in normalized activity, presumably due to the presence of full-length but inactive enzyme in the sample. Because immunodetection does not distinguish between active and inactive UGT, comparison of normalized activity between different batches of a recombinant UGT, mutants of a given UGT, or different UGTs is prone to large inaccuracies. Such inaccuracies could be reduced by lowering the degree of infection of the insect cells, in combination with careful monitoring of UGT expression. However, the latter requires suitable antibodies for comparing UGT expression levels among preparations, antibodies that are not always available. Poly-His (His-tag)-containing peptides, fused to the UGT C terminus, allow sensitive immunodetection of expressed enzymes with monoclonal antibodies. We have now carefully examined the effects of two such fusion peptides on enzyme kinetics. A minor increase in the K(m) values has been detected in the His-tagged UGTs, but no changes in parameters such as the kinetic model and the effects of albumin addition.

Mutation analysis in UGT1A9 suggests a relationship between substrate and catalytic residues in UDP-glucuronosyltransferases.

UDP-glucuronosyltransferases (UGTs) catalyze the transfer of glucuronic acid from UDP-glucuronic acid to endo- and xenobiotics in our body. UGTs belong to the GT1 family of glycosyltransferases and many GT1s use a serine protease-like catalytic mechanism in which an Asp-His pair deprotonates a hydroxyl on the aglycone for nucleophilic attack on the sugar donor. The pair in human UGTs could be H37 and either D143 or D148 (UGT1A9 numbering). However, H37 is not totally conserved, being replaced by either Pro or Leu in UGT1A4 and UGT2B10. We therefore investigated the role of H37, D143 and D148 in UGT1A9 by site-directed mutagenesis, activity and kinetic measurements with several substrates. The results suggest that H37 is not critical in N-glucuronidation, but is so in O-glucuronidation. The V(max) of the H37A mutant was much less affected in N- than O-glucuronidation, while the reverse was true for the Asp mutations, particularly D143A. We suggest that this is due to the opposing properties of O- and N- nucleophiles. O-nucleophiles require the histidine to deprotonate them so that they become effective nucleophiles, while N-nucleophiles develop a formal positive charge during the reaction (RNH(2)(+)-GlcA), and thus require a negatively charged residue to stabilize the transition state.

The first aspartic acid of the DQxD motif for human UDP-glucuronosyltransferase 1A10 interacts with UDP-glucuronic acid during catalysis.

All UDP-glucuronosyltransferase enzymes (UGTs) share a common cofactor, UDP-glucuronic acid (UDP-GlcUA). The binding site for UDP-GlcUA is localized to the C-terminal domain of UGTs on the basis of amino acid sequence homology analysis and crystal structures of glycosyltransferases, including the C-terminal domain of human UGT2B7. We hypothesized that the (393)DQMDNAK(399) region of human UGT1A10 interacts with the glucuronic acid moiety of UDP-GlcUA. Using site-directed mutagenesis and enzymatic analysis, we demonstrated that the D393A mutation abolished the glucuronidation activity of UGT1A10 toward all substrates. The effects of the alanine mutation at Q(394),D(396), and K(399) on glucuronidation activities were substrate-dependent. Previously, we examined the importance of these residues in UGT2B7. Although D(393) (D(398) in UGT2B7) is similarly critical for UDP-GlcUA binding in both enzymes, the effects of Q(394) (Q(399) in UGT2B7) to Ala mutation on activity were significant but different between UGT1A10 and UGT2B7. A model of the UDP-GlcUA binding site suggests that the contribution of other residues to cosubstrate binding may explain these differences between UGT1A10 and UGT2B7. We thus postulate that D(393) is critical for the binding of glucuronic acid and that proximal residues, e.g., Q(394) (Q(399) in UGT2B7), play a subtle role in cosubstrate binding in UGT1A10 and UGT2B7. Hence, this study provides important new information needed for the identification and understanding of the binding sites of UGTs, a major step forward in elucidating their molecular mechanism.

The human UDP-glucuronosyltransferase: identification of key residues within the nucleotide-sugar binding site.

UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism, detoxification,and clearance of many different xenobiotics, including drugs and endogenous compounds. Structural information about these membrane-bound enzymes of the endoplasmic reticulum is limited. We do not know the identity or the location of the key residues for catalysis and binding of the aglycone substrate and the cosubstrate UDP-glucuronic acid (UDPGA). One suggestion was that His371 (UGT1A6 numbering) is the "catalytic base" that deprotonates the phenol group. We have now re-examined this hypothesis by analyzing the activities of the corresponding mutants, 6H371A (in UGT1A6) and the 9H369A (in UGT1A9). The K(m) values of mutant 6H371A for scopoletin and UDPGA were higher by 4- and 11-fold, respectively, than in UGT1A6. The K(d) for the enzyme-UDPGA complex, derived from bisubstrate kinetics, was about 9-fold higher in 6H371A than in UGT1A6, indicating severely impaired cosubstrate binding by the mutant. The effect of mutation on V(max) was large in UGT1A6 but variable in UGT1A9, suggesting that His371 does not play the catalytic role previously hypothesized. In both UGTs, the E379A mutation (UGT1A6 numbering) had an effect similar to that of the H371A mutations. A homology model of the putative UDPGA binding region of UGT1A6 was built using distant homologous protein structures from the "GT1 class." The combined results of activity determinations, kinetic analyses, and modeling strongly suggest that His371 and Glu379 are directly involved in UDPGA binding but are not the general acid or general base.

Interactions with other human UDP-glucuronosyltransferases attenuate the consequences of the Y485D mutation on the activity and substrate affinity of UGT1A6.

OBJECTIVES: To explore the possible role of hetero-oligomerization among the human UDP-glucuronosyltransferases in attenuating the consequences of the pathological Y486D mutation (UGT1A1 numbering) that often causes hyperbilirubinaemia. Owing to exon sharing in the human UGT1A gene, the equivalent mutation is present in all other UGT1As of the affected individuals. It is unknown, however, if this mutation results in clinical conditions, other than impaired bilirubin conjugation by UGT1A1. METHODS: The main experimental approach in this study was to try and form hetero-oligomers of selected UDP-glucuronosyltransferases by coinfecting insect cells with recombinant baculoviruses that encode different human UDP-glucuronosyltransferases and mutants thereof. The infected cells were analysed for both relative expression levels and catalytic activity in each case, the combination of which yielded normalized activity. Kinetic analyses and copurification by affinity chromatography were also performed. RESULTS: Coinfections with UGT1A4 increased the normalized scopoletin glucuronidation of 6YD (the Y485D mutant of UGT1A6) much more than it affected 1YD (the Y486D mutant of UGT1A1). Serotonin glucuronidation analyses revealed that coexpression of 6YD with most other human UDP-glucuronosyltransferases significantly increased the normalized activity of this mutant. Using 1-naphthol as the aglycone substrate, the Km of 6YD for the cosubstrate UDP-glucuronic acid was about 50 times higher than in UGT1A6. Yet, coexpression of 6YD with UGT1A4 lowered the Km for UDP-glucuronic acid to the level of UGT1A6. Coexpression also influenced wild-type UGT1A6 and UGT2B7, increasing the normalized activity of UGT1A6, but decreasing it for UGT2B7. CONCLUSION: Hetero-oligomerization may play an important role in UDP-glucuronosyltransferases activity.