A critical amino acid residue, Asp446, in UDP-glucuronosyltransferase

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Abstract
An amino acid residue, Asp446, 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, A1337G and G1384A (named Ysh A1337GC1384A), that result in two amino acid substitutions, D446G and V462M, 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 A1337GG1384A had no transferase activity. Two other mutant cDNAs with Ysh A1337G having one changed base, A1337G, resulting in one amino acid substitution, D446G, and Ysh G1384A having a changed base, G1384A, resulting in an amino acid substitution, V462M, were constructed and expressed in the yeast. The expressed protein from Ysh G1384A (named Ysh V462M) exhibited enzymic activity, but the one from Ysh A1337G (named Ysh D446G) did not show any activity at all. Asp446 was conserved in all UGTs and UDP-galactose:ceramide galactosyltransferases reported, suggesting that Asp446 plays a critical role in each enzyme.