The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation

Abstract

Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the K(m) value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K(m) values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K(m) in trans-resveratrol, and V(max) and K(si) in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.

Comprehensive Characterization of Mouse UDP-Glucuronosyltransferase (Ugt) Belonging to the Ugt2b Subfamily: Identification of Ugt2b36 as the Predominant Isoform Involved in Morphine Glucuronidation

“…A number of reports have described changes in the level of mouse Ugt isoforms, depending on gender, disease, or chemicals Klaassen, 2007, 2009a,b). Although there is a study of the substrate specificity of mouse Ugt1a6a and 1a6b (Uchihashi et al, 2012), the substrate specificities of all mouse Ugts, except Ugt1a6, remain to be clarified. In this study, we comprehensively characterized the substrate specificity of Ugt1a1, Ugt2a3, and all the Ugt2b subfamily isoforms expressed in the mouse liver.…”

“…Many studies have investigated changes in the expression level of Ugt isoforms in mice, involving disease, chemicals, or genetically engineered model animals (Buckley and Klaassen, 2007;2009a,b;Lu et al, 2010). Furthermore, knocking out the Ugt2 locus in mice has been reported and characterized (Fay et al, 2015); however, characterization of mouse Ugt isoforms, except 1a6 (Uchihashi et al, 2012(Uchihashi et al, , 2013, has not been reported, and it would be of great interest to understand the function of mouse Ugts, especially the Ugt2b subfamily.…”

Biosynthesis of Drug Glucuronide Metabolites in the Budding Yeast Saccharomyces cerevisiae

“…Several lines of evidence indicated that budding yeast, as host cells, could be used for the expression of UGT enzymes by employing the pGYR vector with a yeast glyceraldehyde 3-dehydrogenase promoter and terminator. [14][15][16][17]25 When expressing human UGTs in budding yeast, some of them, particularly UGT1A4, exhibited lower expression level than others (Figure 1). We found that replacing the signal peptide of the low-level expressed UGTs with the signal peptide of the human UGT1A7 stimulated their expression and led to nearly similar expression levels for all the human UGT1As (Figure 1).…”

“…The yeast ( Saccharomyces cerevisiae ) strain AH22 was used for protein expression, as previously reported. − Since the genome-integrating vector pAUR has an aureobasidin A-resistant gene, AH22 transformants with pAUR are able to grow in YPD medium (1% (w/v) yeast extract, 2% (w/v) polypeptone, 2% (w/v) glucose), supplemented with 0.5 μg/mL aureobasidin A (Takara, Otsu, Japan). The expression plasmids were digested by BsiWI prior to transformation (0.5–1 μg DNA/10 μL, 10 μL), which was done using the lithium chloride method .…”

“…Budding yeast cells were previously used for heterologous expression of UGTs. − Nevertheless, due to their inability to generate UDPGA, resting budding yeast cells are unable to convert drugs into glucuronides using the expressed UGTs. The latter was changed, however, when heterologous expression of plant UGDH enabling budding yeast to produce UDP-glucuronic acid by de novo synthesis from intrinsic UDP-glucose .…”