17238-47-0Relevant articles and documents
Critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-glucuronosyltransferases 1A3 and 1A4
Kubota, Takahiro,Lewis, Benjamin C.,Elliot, David J.,Mackenzie, Peter I.,Miners, John O.
, p. 1054 - 1062 (2007)
Despite high sequence identity, UGT1A3 and UGT1A4 differ in terms of substrate selectivity. UGT1A3 glucuronidates the planar phenols 1-naphthol (1-NP) and 4-methylumbelliferone (4-MU), whereas UGT1A4 converts the tertiary amines lamotrigine (LTG) and trifluoperazine (TFP) to quaternary ammonium glucuronides. Residues 45 to 154 (which incorporate 21 of the 35 amino acid differences) and 45 to 535 were exchanged between UGT1A3 and UGT1A4 to generate UGT1A3-4(45-535), UGT1A3-4(45-154)-3, UGT1A4-3 (45-535), and UGT1A4-3(45-154)-4 hybrid proteins. Although differences in kinetic parameters were observed between the parent enzymes and chimeras, UGT1A4-3(45-535) and UGT1A4-3(45-154)-4 [but not UGT1A3-4(45-535) and UGT1A3-4(45-154)-3] retained the capacity to glucuronidate LTG and TFP. Likewise, UGT1A3-4(45-535) and UGT1A3-4(45-154)-3 retained the capacity to glucuronidate 1-NP and 4-MU, but UGT1A4-3(45-535) and UGT1A4-3(45-154)-4 exhibited low or absent activity. Within the first 44 residues, UGT1A3 and UGT1A4 differ in sequence at positions 36 and 40. "Reciprocal" mutagenesis was performed to generate the UGT1A3(I36T), UGT1A3(H40P), UGT1A4(T36I), and UGT1A4 (P40H) mutants. The T36I and P40H mutations in UGT1A4 reduced in vitro clearances for LTG and TFP glucuronidation by >90%. Conversely, the I36T and H40P mutations in UGT1A3 reduced the in vitro clearances for 1-NP and 4-MU glucuronidation by >90%. Introduction of the single H40P mutation in UGT1A3 conferred LTG and TFP glucuronidation, whereas the single T36I mutation in UGT1A4 conferred 1-NP and 4-MU glucuronidation. Thus, residues 36 and 40 of UGT1A3 and UGT1A4 are pivotal for the respective selectivities of these enzymes toward planar phenols and tertiary amines, although other regions of the proteins influence binding affinity and/or turnover. Copyright
Phenylalanine 93 of the human UGT1A10 plays a major role in the interactions of the enzyme with estrogens
H?glund, Camilla,Sneitz, Nina,Radominska-Pandya, Anna,Laakonen, Liisa,Finel, Moshe
experimental part, p. 1465 - 1473 (2011/11/29)
Little is currently known about the substrate binding site of the human UDP-glucuronosyltransferases (UGTs) and the structural elements that affect their complex substrate selectivity. In order to further understand and extend our earlier findings with phenylalanines 90 and 93 of UGT1A10, we have replaced each of them with Gly, Ala, Val, Leu, Ile or Tyr, and tested the activity of the resulting 12 mutants toward eight different substrates. Apart from scopoletin glucuronidation, the F90 mutants other than F90L were nearly inactive, while the F93 mutants' activity was strongly substrate dependent. Hence, F93L displayed high entacapone and 1-naphthol glucuronidation rates, whereas F93G, which was nearly inactive in entacapone glucuronidation, was highly active toward estradiol, estriol and even ethinylestradiol, a synthetic estrogen that is a poor substrate for the wild-type UGT1A10. Kinetic analyses of 4-nitrophenol, estradiol and ethinylestradiol glucuronidation by the mutants that catalyzed the respective reactions at considerable rates, revealed increased Km values for 4-nitrophenol and estradiol in all the mutants, whilst the K m values of F93G and F93A for ethinylestradiol were lower than in control UGT1A10. Based on the activity results and a new molecular model of UGT1A10, it is suggested that both F90 and F93 are located in a surface helix at the far end of the substrate binding site. Nevertheless, only F93 directly affects the selectivity of UGT1A10 toward large and rigid estrogens, particularly those with substitutions at the D ring. The effects of F93 mutations on the glucuronidation of smaller or less rigid substrates are indirect, however.
