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Research ArticleArticle

The Human UDP-Glucuronosyltransferase: Identification of Key Residues within the Nucleotide-Sugar Binding Site

Anne-Sisko Patana, Mika Kurkela, Adrian Goldman and Moshe Finel
Molecular Pharmacology September 2007, 72 (3) 604-611; DOI: https://doi.org/10.1124/mol.107.036871
Anne-Sisko Patana
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Mika Kurkela
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Adrian Goldman
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Moshe Finel
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Abstract

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 Km values of mutant 6H371A for scopoletin and UDPGA were higher by 4- and 11-fold, respectively, than in UGT1A6. The Kd 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 Vmax 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.

Footnotes

  • This work was supported by the Academy of Finland (A.G. projects 1105157 and 1114752, M.F. projects 207535 and 210933) and the Sigrid Juselius Foundation (A.G. and M.F.). A.G. is a member of Biocentrum Helsinki, which partly supported this work.

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

  • doi:10.1124/mol.107.036871.

  • ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; UDPGA, UDP-glucuronic acid; ER, endoplasmic reticulum; 4-MU, 4-methylumbelliferone; GT, glycosyltransferase; CAZY, carbohydrate active enzymes; PDB, Protein Data Bank; UGT71G1, triterpene/flavonoid glycosyltransferase from legume Medicago truncatula; VvGT, UDP-glucose:flavonoid 3-O-glycosyltransferase from red grape (Vitis vinifera); GtfA, TDP-epi-vancosaminyl-transferase from Amycolatopsis orientalis; GtfB, UDP-glucosyltransferase from Amycolatopsis orientalis; GtfD, vancosaminyltransferase from Amycolatopsis orientalis; MurG UDP-N-acetylglucosaminyltransferase from Escherichia coli; UGT1A6, human UDP-glucuronosyltransferase isoenzyme 1A6; UGT1A9, human UDP-glucuronosyltransferase isoenzyme UGT1A9.

    • Received April 5, 2007.
    • Accepted June 19, 2007.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 72 (3)
Molecular Pharmacology
Vol. 72, Issue 3
1 Sep 2007
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Research ArticleArticle

The Human UDP-Glucuronosyltransferase: Identification of Key Residues within the Nucleotide-Sugar Binding Site

Anne-Sisko Patana, Mika Kurkela, Adrian Goldman and Moshe Finel
Molecular Pharmacology September 1, 2007, 72 (3) 604-611; DOI: https://doi.org/10.1124/mol.107.036871

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Research ArticleArticle

The Human UDP-Glucuronosyltransferase: Identification of Key Residues within the Nucleotide-Sugar Binding Site

Anne-Sisko Patana, Mika Kurkela, Adrian Goldman and Moshe Finel
Molecular Pharmacology September 1, 2007, 72 (3) 604-611; DOI: https://doi.org/10.1124/mol.107.036871
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