An active and water-soluble truncation mutant of the human UDP-glucuronosyltransferase 1A9.
The UDP-glucuronosyltransferases (UGTs) are integral membrane proteins, and previous attempts to generate a water-soluble UGT by removing the single trans-membrane helix yielded inactive and membrane-bound proteins. We have now replaced the 45 C-terminal amino acids of the human UGT1A9, including its trans-membrane helix, with a fusion peptide ending with six His residues. Detergent-free extraction of insect cells expressing this mutant, UGT1A9Sol, released scopoletin glucuronidation activity into the supernatant, and subsequent ultracentrifugation did not sediment that activity. UGT1A9Sol was purified by immobilized metal affinity chromatography (IMAC) in the absence of detergents throughout the entire process. The IMAC purification increased somewhat the apparent K(m) of UGT1A9 toward scopoletin and rendered the enzyme sensitive to freezing. The activity of UGT1A9Sol in the cell extract was partly inhibited by Triton X-100, irrespective of the presence or absence of phospholipids. UGT1A9Sol exhibited a relatively high rate of scopoletin glucuronidation, whereas its activity toward 1-naphthol, entacapone, umbelliferone, and 4-nitrophenol was much lower. The kinetics and substrate specificity of UGT1A9Sol resembled the detergent-suspended full-length UGT1A9 rather than the membrane-bound UGT1A9. The apparent K(m) value of UGT1A9Sol for scopoletin was similar to that of the full-length UGT1A9 in the presence of detergent, but much higher than the respective value in the membrane-bound enzyme. The results suggest that either the detergent binding to the trans-membrane helix within the full-length UGT1A9, or the removal of this helix by gene manipulation, affect the interaction of the enzyme with its aglycone substrate in a similar manner.
Kurkela
Mika
M
Viikki Drug Discovery Technology Center, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland.
Mörsky
Saila
S
Hirvonen
Jouni
J
Kostiainen
Risto
R
Finel
Moshe
M
D017353:Gene Deletion; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D017434:Protein Structure, Tertiary; D012995:Solubility; D013379:Substrate Specificity; D000090264:UDP-Glucuronosyltransferase 1A9; D014867:Water / Q000737:chemistry
C000715591:UGT1A9 protein, human; D014867:Water; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9