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[摘要]：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 values for HFC glucuronidation of UGT1A8, UGT1A9 and their chimeras were divided into two types, UGT1A8 type (high K and UGT1A9 type (low K-m), and these types were determined according to whether their amino acids at positions 69132 were those of UGT1A8 or UGT1A9. Likewise, PB O-glucuronidating activity was also detected by all of the chimeras, and their K,,, 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. (c) 2008 Elsevier Inc. All rights reserved. |
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