Essential role of residue H49 for activity of Escherichia coli 1-deoxy-D-xylulose 5-phosphate synthase, the enzyme catalyzing the first step of the 2-C-methyl-D-erythritol 4-phosphate pathway for isoprenoid synthesis

The first step of the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for isoprenoid biosynthesis in plant plastids and most eubacteria is catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), a recently described tra nsketolase-like enzyme. To identify key residues for DXS activity, we compa red the amino acid sequence of Escherichia coli DXS with that of E. coli an d yeast transketolase (TK). Alignment showed a previously undetected conser ved region containing an invariant histidine residue that has been describe d to participate in proton transfer during TK catalysis. The possible role of the conserved residue in E. coli DXS (H49) was examined by site-directed mutagenesis. Replacement of this histidine residue with glutamine yielded a mutant DXS-H49Q enzyme that showed no detectable DXS activity. These find ings are consistent with those obtained for yeast TK and demonstrate a key role of H49 for DXS activity. (C) 2001 Academic Press.