RESUMO
The inhibition of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) by fosmidomycin was studied by using a kinetic assay based on the consumption of NADPH and synthetic substrate. Fosmidomycin is a slow tight-binding inhibitor of DXR that shows strong negative cooperativity (absolute value(h) = 0.3) in binding. Cooperativity is displayed during the initial (weak, K0.5 = 10 microM) binding event and does not change as the binding tightens to the equilibrium value of 0.9 nM over a period of seconds to minutes. A series of fosmidomycin fragments was examined, but all showed much weaker inhibition, in the mM range. A series of cyclic fosmidomycin analogues was also synthesised and tested, but these showed high-microM binding at best. None of the synthetic compounds showed time-dependent inhibition. We concluded that the slow tight-binding behaviour, and perhaps also cooperativity, are mediated by significant reorganisation of the active site upon fosmidomycin binding. This makes the rational design of new inhibitors of DXR difficult at best.
Assuntos
Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/química , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Fosfomicina/análogos & derivados , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Aldose-Cetose Isomerases/metabolismo , Antibacterianos/química , Sítios de Ligação , Inibidores Enzimáticos/química , Escherichia coli/enzimologia , Fosfomicina/síntese química , Fosfomicina/química , Fosfomicina/farmacologia , Cinética , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Oxirredutases/metabolismoRESUMO
1-Deoxy-D-xylulose-5-phosphate (DXP) is a key intermediate in the non-mevalonate pathway to terpenoids in bacteria, and it is the substrate for the enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXP-R). In order to study the mechanism of DXP-R, we required a flexible synthesis of the substrate which would allow the incorporation of isotopic labels, and the variation of the two stereocentres. Thus 1,4-dihydroxypent-2-yne was selectively reduced to give the E-olefin, and selective phosphorylation of the primary alcohol followed by oxidation of the secondary alcohol gave a substrate suitable for dihydroxylation. Dihydroxylation using stoichiometric OsO4 in the presence of chiral ligands gave protected DXP in high ee. Final hydrogenolysis gave DXP in quantitative yield and high purity. DXP-R was produced by rapid cloning of the dxr gene from Escherichia coli through controlled expression and ion exchange chromatography. The synthetic DXP was fully active in enzyme assays catalysed by recombinant DXP-R.
