Amino acid positions 69-132 of UGT1A9 are involved in the C-glucuronidation of phenylbutazone.
Phenylbutazone (PB) is known to be biotransformed to its O- and C-glucuronide. Recently, we reported that PB C-glucuronide formation is catalyzed by UGT1A9. Interestingly, despite UGT1A8 sharing high amino acid sequence identity with UGT1A9, UGT1A8 had no PB C-glucuronidating activity. In the present study, we constructed eight UGT1A9/UGT1A8 chimeras and evaluated which region is important for PB C-glucuronide formation. All of the chimeras and UGT1A8 and UGT1A9 had 7-hydroxy-(4-trifluoromethyl)coumarin (HFC) O-glucuronidating activity. The K(m) values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K(m)) and UGT1A9 type (low K(m)), and these types were determined according to whether their amino acids at positions 69-132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K(m) values were divided into two types. On the contrary, PB C-glucuronidating activity was detected by UGT1A9((1-132))/1A8((133-286)), UGT1A9((1-212))/1A8((213-286)), UGT1A8((1-68))/1A9((69-286)), and UGT1A8((1-68))/1A9((69-132))/1A8((133-286)) chimeras. The region 1A9((69-132)) was common among chimeras having PB C-glucuronidating activity. Of interest is that UGT1A9((1-68))/1A8((69-132))/1A9((133-286)) had lost PB C-glucuronidation activity, but retained activities of PB and HFC O-glucuronidation. These results strongly suggested that amino acid positions 69-132 of UGT1A9 are responsible for chemoselectivity for PB and affinity to substrates such as PB and HFC.
Nishiyama
Takahito
T
Department of Drug Metabolism and Molecular Toxicology, School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji-shi, Tokyo 192-0392, Japan.
Fujishima
Miki
M
Masuda
Yasuhiro
Y
Izawa
Tadashi
T
Ohnuma
Tomokazu
T
Ogura
Kenichiro
K
Hiratsuka
Akira
A
D000596:Amino Acids / Q000737:chemistry*; D002384:Catalysis; D003374:Coumarins / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D014453:Glucuronosyltransferase / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D010653:Phenylbutazone / Q000737:chemistry* / Q000494:pharmacology; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D000090264:UDP-Glucuronosyltransferase 1A9
D000596:Amino Acids; D003374:Coumarins; D017931:DNA Primers; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; C000715591:UGT1A9 protein, human; C056033:7-ethoxy-4-trifluoromethylcoumarin; D014453:Glucuronosyltransferase; D000090264:UDP-Glucuronosyltransferase 1A9; C454320:UDP-glucuronosyltransferase, UGT1A8; D010653:Phenylbutazone