Analysis of active-site residues in hyoscyamine 6β-hydroxylase

Abstract

Three amino acid residues (histidine-217, aspartic acid-219, and histidine-274) in Hyoscyamus niger hyoscyamine 6β-hydroxylase are strictly conserved among 2-oxoglutarate-dependent dioxygenases and other structurally related enzymes. These residues were investigated by chemical modification and site-directed mutagenesis. The hydroxylase was expressed in E. coli as a fusion protein to maltose-binding protein. Modification of histidine residues by diethyl pyrocarbonate inactivated the recombinant wild-type hydroxylase. Inactivation was prevented most effectively by the presence of 2-oxoglutarate. Mutation of histidine217 to glutamine, histidine-274 to glutamine, or aspartic acid-219 to either histidine or asparagine inactivated the hydroxylase, whereas substitution of loosely conserved histidine-66 with glutamine did not decrease the catalytic activity of the enzyme. These results suggest that histidine-217, aspartic acid-219, and histidine274 play important roles in the hydroxylase function, and may be the ligands to the active-site iron.