REQUIRED BURIED ALPHA-HELICAL STRUCTURE IN THE BILIRUBIN UDP-GLUCURONOSYLTRANSFERASE, UCT1A1, CONTAINS NONREPLACEABLE PHENYLALANINE

A conserved hydrophobic region in the bilirubin-type UDP-glucuronosylt ransferase isozyme was first uncovered as a consequence of a deleterio us mutation in the UGT1A1 (HUG-Br1) isozyme of a Crigler-Najjar (CN) T ype I patient. According to analysis by the RAOARGOS computer program, this hydrophobic region in UGT1A1 is located between residues 159-177 and defines a buried helix centered over position 169-172 with a posi tive factor of 1.22. Further analysis showed that the planar phenol-ty pe UGT1A6 (HLUG P1) isoform, unlike the steroid-type UGT2B7 (UDPGTh2) isozyme, has a similar conserved hydrophobic region and that the posit ive factor for its buried helix is 1.14 compared to the threshold of 1 .13 for such a structure. The analysis detected the typical membrane-i nsertion-signal sequence and a membrane-anchoring domain in each isofo rm. The different amino acid sequence patterns between positions 168-1 72 for the three types of isoforms and the deleterious mutations in th is microregion (MRA) of UGT1A1 in CN-I patients are evidence of a crit ical and discriminating role for MRA. With the recombinant UGT1A1 enzy me and its mutants, P167G, F170del, F170L, F170I F170V, F170A, F170Y, F170E, F171L, F171I, F171V, F171A, F171Y, or L175Q, expressed in COS-1 cells, bilirubin glucuronidating activity at both pH 6.4 and 7.6 demo nstrated that Phe-170 is not replaceable, whereas Phe-171 can be repla ced by Leu without any loss of activity. The less hydrophobic buried h elix in the phenolic-type UGT1A6 has a Tyr/Leu at position 170/171; th is isoform glucuronidated bilirubin at 1/10 the level of that by UGT1A 1 with a Km (bilirubin) of 25 mu M compared to that for UGT1A1 of 5.0 mu M.