Methods to find inhibitors of riboflavin biosynthesis, DNA encoding GTP-cyclohydrolase II or 3,4-dihydroxy-2-butanone-4-phosphate synthase and related bifunctional proteins

摘要

Identifying inhibitors of GTP-cyclohydrolase II and GTP (I) or 3,4-dihydroxy-2-butanone-4-phosphate synthase (II), comprising comparing concentrations of 2,5-diamino-6-ribosylamino-2,4(1H,3H)-pyrimidindion-5'-phosphate or 3,4-dihydroxy-2-butanone-4-phosphate before and after test compound addition to a mixture of (I) or (II) and ribulose-5-phosphate. Screening for presence or absence of inhibition of GTP-cyclohydrolase II activity, comprises the following steps: (a) production of a first aqueous mixture with a content of a protein with a GTP-cyclohydrolase II sequence and GTP; (b) transforming the first mixture in question during a predefined time at a predefined temperature and determining the concentration of 2,5-diamino-6-ribosylamino-2,4(1H,3H)-pyrimidindion-5'-phosphate (A); (c) producing a second aqueous mixture by addition of a predetermined quantity of a chemical test sample to the first mixture; (d) transforming the second mixture during a predetermined time at a predefined temperature and determining the concentration of (A); and (e) determining the presence of inhibition of GTP-cyclohydrolase II by establishing if the determined concentration in step (d) is lower than that in step (b). Independent claims are also included for the following: (1) a reagent systems for use in the novel method; (2) an isolated protein with plant enzyme sequence and function of GTP-cyclohydrolase II and/or 3,4-dihydroxy-2-butanone-4-phosphate synthase; (3) isolated DNA, which codes a protein of (2), and optionally at least one other enzyme of the riboflavin biosynthesis pathway; (4) a vector containing a nucleotide sequence for DNA of (3); (5) a method to inhibit enzymes with GTP-cyclohydrolase II and 3,4-dihydroxy-2-butanone-4-phosphate synthase activity for, or in, a plant through treatment with a compound, which is chosen from the group of chemical compounds, which show inhibition in the novel method; and (6) a method to inhibit an enzyme with GTP-cyclohydrolase II or 3,4-dihydroxy-2-butanone-4-phosphate synthase activity from, or in, a microorganism through treatment with a compound, which is chosen from the group of chemical compounds, which show inhibition in the novel method.