Intermediates and enzymes of the non-mevalonate isoprenoid pathway

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.

Cells, cell cultures, or organisms or parts thereof transformed or transfected for an increased rate of formation of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably in its (E)-form, compared to cells, cell cultures, or organisms or parts thereof absent said transformation or transfection.

Cells, cell cultures, or organisms or parts thereof transformed or transfected for an increased rate of conversion of (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate to isopentenyl diphosphate and/or dimethylallyl diphosphate compared to cells, cell cultures, or organisms or parts thereof absent said transformation or transfection.

Optionally isotope-labelled compound of the following formula I or a salt thereof:

• 6
• whereby R1 and R2 are different from each other and one of R1 and R2 is hydrogen and the other is selected from the group consisting of —CH2—O—PO(OH)—O—PO(OH)2, —CH2—O—PO(OH)2, and —CH2OH, and whereby A stands for —CH2OH or —CHO.

Optionally isotope-labelled 1-hydroxy-2-methyl-2-butenyl 4-diphosphate salt or a protonated form thereof.

Optionally isotope-labelled (E)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate salt or a protonated form thereof.

Optionally isotope-labelled (Z)-1-hydroxy-2-methyl-2-butenyl 4-diphosphate salt or a protonated form thereof.

A method of altering the expression level of the gene product(s) of ispG and/or ispH in cells comprising

• (a) transforming host cells with the ispG and/or ispH gene,
• (b) growing the transformed host cells of step (a) under conditions that are suitable for the efficient expression of ispG and/or ispH, resulting in production of altered levels of the ispG and/or ispH gene product(s) in the transformed cells relative to expression levels of untransformed cells.

Method of identifying an inhibitior of an enzyme functional for the conversion of 2C-methyl-D-erythritol 2,4-cyclodiphosphate to 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably its E-form, of the non-mevalonate isoprenoid pathway by the following steps:

• (a) incubating a mixture containing said enzyme with its, optionally isotope-labeled, substrate 2C-methyl-D-erythritol-2,4-cyclodiphosphate 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, and
• (c) comparing the concentration in the presence and in the absence of said potential inhibitor.

Method of identifying an inhibitior of an enzyme functional for the conversion of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate, notably its E-form, to isopentenyl diphosphate or dimethylallyl diphosphate of the non-mevalonate isoprenoid pathway by the following steps:

• (a) incubating a mixture containing said enzyme with its, optionally isotope-labeled, substrate 1-hydroxy-2-methyl-2-butenyl 4-diphosphate under conditions suitable for said conversion in the presence and in the absence of a potential inhibitor,
• (b) determining the concentration of 1-hydroxy-2-methyl-2-butenyl 4-diphosphate and/or isopentenyl diphosphate or dimethylallyl diphosphate, and
• (c) comparing the concentration in the presence and in the absence of said potential inhibitor.