A colorimetric assay for the determination of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase activity.
A new method for the determination of the activity of 4-diphosphocytidyl-2-C-methyl-D-erythritol 4-phosphate synthase, the enzyme catalyzing the third reaction of the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesis of isoprenoids, is described. This is an end-point assay based on the transformation of inorganic pyrophosphate, one of the products of the reaction, to phosphate by using inorganic pyrophosphatase as auxiliary enzyme. The phosphate formed is reacted then with the dye malachite green to yield a colored product which can be determined spectrophotometrically. The method is easy to perform, sensitive, and robust and can be used in automated high-throughput screening analyses for the search of inhibitors of the enzyme.
Bernal
Cristobal
C
Departament de Bioquimica i Biologia Molecular, Universitat de Barcelona, Avda Diagonal 645, 08028 Barcelona, Spain.
Palacin
Celia
C
Boronat
Albert
A
Imperial
Santiago
S
D003124:Colorimetry / Q000379:methods; D004896:Erythritol / Q000031:analogs & derivatives* / Q000032:analysis* / Q000096:biosynthesis / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000032:analysis* / Q000378:metabolism; D043564:Inorganic Pyrophosphatase; D009713:Nucleotidyltransferases / Q000032:analysis* / Q000378:metabolism; D010710:Phosphates; D012394:Rosaniline Dyes; D013057:Spectrum Analysis
C400182:4-diphosphocytidyl-2-C-methylerythritol; D029968:Escherichia coli Proteins; D010710:Phosphates; D012394:Rosaniline Dyes; C005095:malachite green; C400183:4-diphosphocytidyl-2-C--methylerythritol synthase; D009713:Nucleotidyltransferases; D043564:Inorganic Pyrophosphatase; D004896:Erythritol