Critical roles of residues 36 and 40 in the phenol and tertiary amine aglycone substrate selectivities of UDP-glucuronosyltransferases 1A3 and 1A4.

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.