Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Abstract

1-deoxy-D-xylulose 5-phosphate synthase\r(DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate\r(MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived\rfrom pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield\r1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49,\rE370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis\rand kinetic analysis of the purified recombinant enzyme mutants. Mutants at\rposition H49 showed a severe reduction in their specific activities with a\rdecrease of the kcat/KM ratio by two orders of magnitude lower than the\rwild-type DXS. According to available structural data residue H49 is perfectly\rpositioned to abstract a proton from the donor substrate. Mutations in DXS E370\rshowed that this residue is also essential for catalytic activity.\rThree-dimensional structure supports its involvement in cofactor deprotonation,\rthe first step in enzymatic thiamin catalysis. Results obtained with H431\rmutant enzymes indicate that this residue plays a role contributing to\rtransition state stabilization. Finally, mutants at position D427 also showed a\rsevere specific activity decrease with a reduction of the kcat/KM ratio. A role\rin binding the substrate and selecting the stereoisomer is proposed for D427.