1-deoxy-D-xylulose 5-phosphate synthase
(DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate
(MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived
from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield
1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49,
E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis
and kinetic analysis of the purified recombinant enzyme mutants. Mutants at
position H49 showed a severe reduction in their specific activities with a
decrease of the kcat/KM ratio by two orders of magnitude lower than the
wild-type DXS. According to available structural data residue H49 is perfectly
positioned to abstract a proton from the donor substrate. Mutations in DXS E370
showed that this residue is also essential for catalytic activity.
Three-dimensional structure supports its involvement in cofactor deprotonation,
the first step in enzymatic thiamin catalysis. Results obtained with H431
mutant enzymes indicate that this residue plays a role contributing to
transition state stabilization. Finally, mutants at position D427 also showed a
severe specific activity decrease with a reduction of the kcat/KM ratio. A role
in binding the substrate and selecting the stereoisomer is proposed for D427.
Cite this paper
Querol-Audí, J. , Boronat, A. , Centelles, J. and Imperial, S. (2014) Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase. Journal of Biosciences and Medicines
, 30-35. doi: 10.4236/jbm.2014.24006
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