Characterization of 1-Deoxy-d-xylulose 5-Phosphate Reductoisomerase, an Enzyme Involved in Isopentenyl Diphosphate Biosynthesis, and Identification of Its Catalytic Amino Acid Residues

Tomohisa Kuzuyama, Shunji Takahashi, Motoki Takagi, Haruo Seto
2000 Journal of Biological Chemistry  
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 alternative 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 analysis of the dxr genes from 10 E. coli
more » ... ient mutants revealed base substitution mutations at four points: two nonsense mutations and two amino acid substitutions (Gly 14 to Asp 14 and Glu 231 to Lys 231 ). Diethyl 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 except for G14D were obtained as soluble proteins. Although the purified enzyme 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 H257Q for DXP increased to 3.5-, 7.6-, and 19-fold the wild-type value, respectively. These results indicate that Glu 231 of E. coli DXP reductoisomerase plays an important role(s) in the conversion of DXP to 2-C-methyl-D-erythritol 4-phosphate, and that His 153 , His 209 , and His 257 , in part, associate with DXP binding in the enzyme molecule.
doi:10.1074/jbc.m001820200 pmid:10787409 fatcat:unoa7ayumnbwhftowcsuwtwj5m