Characterization of 1-Deoxy-D-xylulose 5-phosphate reductoisomerase, an enzyme involved in isopentenyl diphosphate biosynthesis, and identification of its catalytic amino acid residues

1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase, which simultaneously catalyzes the intramolecular rearrangement and reduction of DXP to form 2- C-methyl-D-erythritol 4-phosphate, constitutes a key enzyme of an alternati ve mevalonate-independent pathway for isopentenyl diphosphate biosynthesis. The dxr gene encoding this enzyme from Escherichia coli was overexpressed as a histidine-tagged protein and characterized in detail. DNA sequencing a nalysis of the dxr genes from 10 E. coli dxr-deficient mutants revealed bas e substitution mutations at four points: two nonsense mutations and two ami no acid substitutions (Gly(14) to Asp(14) and Glu(231) to Lys(231)), Diethy l pyrocarbonate treatment inactivated DXP reductoisomerase, and subsequent hydroxylamine treatment restored the activity of the diethyl pyrocarbonate- treated enzyme. To characterize these defects, we overexpressed the mutant enzymes G14D, E231K, H153Q, H209Q, and H257Q. All of these mutant enzymes e xcept for G14D were obtained as soluble proteins. Although the purified enz yme E231K had wildtype K-m values for DXP and NADPH, the mutant enzyme had less than a 0.24% wild-type k(cat) value. K-m values of H153Q, H209Q, and H 257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, resp ectively. These results indicate that Glu(231) of E. coli DXP reductoisomer ase plays an important role(s) in the conversion of DXP to 2-C-methyl-D-ery thritol 4-phosphate, and that His(153), His(209), and His(257), in part, as sociate with DXP binding in the enzyme molecule.