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Altincicek, B., Hintz, M., Sanderbrand, S., Wiesner, J., Beck, E. and Jomaa, H. (2000) Tools for Discovery of Inhibi- tors of the 1-Deoxy-D-Xylulose 5-Phosphate (DXP) Synthase and DXP Reductoisomerase: An Approach with En- zymes from the Pathogenic Bacterium Pseudomonas aeruginosa. FEMS Microbiology Letters, 190, 329-333.

has been cited by the following article:

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

    AUTHORS: Jordi Querol-Audí, Albert Boronat, Josep J. Centelles, Santiago Imperial

    KEYWORDS: Active Site, 1-Deoxy-D-Xylulose 5-Phosphate Synthase, Isoprenoid Biosynthesis, Kinetic Parameters, MEP Pathway, Methylerythritol Phosphate, Mutagenesis

    JOURNAL NAME: Journal of Biosciences and Medicines, Vol.2 No.4, June 12, 2014

    ABSTRACT: 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.