A colorimetric assay for the determination of 4-diphosphocytidyl-2- C-methyl- d-erythritol 4-phosphate synthase activity

• Published in: Analytical biochemistry Vol. 337; no. 1; pp. 55 - 61
• Main Authors: Bernal, Cristobal, Palacin, Celia, Boronat, Albert, Imperial, Santiago
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.02.2005
• Subjects: 2- C-Methyl- d-erythritol 4-phosphate pathway; 4-Diphosphocytidyl-2- C-methyl- d-erythritol 4-phosphate synthase; Colorimetric assay; Colorimetry - methods; Erythritol - analogs & derivatives; Erythritol - analysis; Erythritol - biosynthesis; Erythritol - metabolism; Escherichia coli Proteins - analysis; Escherichia coli Proteins - metabolism; Inorganic Pyrophosphatase; Isoprenoid biosynthesis; Malachite green; Nucleotidyltransferases - analysis; Nucleotidyltransferases - metabolism; Phosphates; Pyrophosphatase; Rosaniline Dyes; Spectrum Analysis; 2- C-Methyl- d-erythritol 4-phosphate pathway; Colorimetric assay; 4-Diphosphocytidyl-2- C-methyl- d-erythritol 4-phosphate synthase; Pyrophosphatase; Isoprenoid biosynthesis; Malachite green
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2004.10.011

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.