Intermediates and enzymes of the non-mevalonate isoprenoid pathway
View Patent ↗The invention provides a protein in a form that is functional for the enzymatic conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate notably in its (E)-form of the non-mevalonate biosynthetic pathway to isoprenoids. The invention also provides a protein in a form that is functional for the enzymatic conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably in its (E)-form, to isopentenyl diphosphate and/or dimethylallyl diphosphate. Further, screening methods for inhibitors of these proteins are provided. Further, 1-hydroxy-2-methyl-2-butenyl 4-diphosphate is provided and chemical and enzymatic methods of its preparation.
1. A method of identifying an inhibitor of an enzyme functional for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, in its E-form, of the non-mevalonate isoprenoid pathway comprising:
(a) incubating a mixture containing said enzyme with its, optionally isotope labeled, substrate 2C-methyl-D-erythritol 2,4-cyclodiphosphate and NADH or NADPH under conditions suitable for said conversion in the presence and in the absence of a potential inhibitor,
(b) subsequently determining the concentration of 2C-methyl-D-erythritol 2,4-cyclodiphosphate and/or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate by measuring the consumption of NADH or NADPH, and
(c) comparing the concentration of 2C-methyl-D-erythritol 2,4-cyclodiphosphate and/or 1-hydroxy-2-methyl-2-butenyl 4-diphosphate in the presence and in the absence of said potential inhibitor.
2. The method according to claim 1 , wherein said enzyme is a plant enzyme.
3. The method according to according to claims 1 , wherein said enzyme is an enzyme of Plasmodium falciparum.
4. The method according to according to claim 1 , wherein said enzyme is a bacterial enzyme.
5. The method according to according to claim 1 , wherein the incubation of step (a) is carried out in the presence of a sulfhydryl reductant.
6. The method according to according to claim 1 , wherein the incubation in step (a) is carried out in the presence of a phosphatase inhibitor.
7. The method according to claim 6 , wherein the phosphatase inhibitor is an alkali fluoride.
8. The method according to claim 1 , wherein the incubation in step (a) is carried out in the presence of pamidronate.
9. The method according to claim 1 , wherein the incubation of step (a) is carried out in the presence of a salt selected from the group of Co 2+ , Mn 2+ , Fe 2+ , Ni 2+ salts.
10. The method according to claim 1 , wherein step (b) is carried out by measuring the consumption of NADH or NADPH by absorbance and/or fluorescence spectrophotometry.
11. The method according to claim 1 further comprising simultaneously or consecutively performing steps (a) through (c) with at least one other potential inhibitor.
12. The method according to claim 5 , wherein the sulfhydryl reductant is dithiothreitol.