A critical amino acid residue, Asp(446), in UDP-glucuronosyltransferase

An amino acid residue, Asp(446), was found to be essential for the enzymic activity of UDP-glucuronosyltransferase (UGT). We obtained a rat phenol UGT (UGT1*06) cDNA (named Ysh) from male rat liver by reverse-transcription (RT)-PCR using pfu polymerase. A mutant Ysh having two different bases, A(1337)G and G(1384)A (named Ysh A(1337)GC(1384)A), that result in two amino acid substitutions, D(446)G and (VM)-M-462, was obtained by RT-PCR using Taq polymerase. Ysh was expressed functionally in microsomes of Saccharomyces cerevisiae strain AH22. However, the expressed protein from Ysh A(1337)GG(1384)A had no transferase activity. Two other mutant cDNAs with Ysh A(1337)G having one changed base, A(1337)G, resulting in one amino acid substitution, D(446)G, and Ysh G(1384)A having a changed base, G(1384)A, resulting in an amino acid substitution, (VM)-M-462, were constructed and expressed in the yeast. The expressed protein from Ysh G(1384)A (named Ysh (VM)-M-462) exhibited enzymic activity, but the one from Ysh A(1337)G (named Ysh D(446)G) did not show any activity at all. Asp(446) was conserved in all UGTs and UDP-galactose: ceramide galactosyltransferases reported, suggesting that Asp(446) plays a critical role in each enzyme.