EP1141381A1

EP1141381A1 - Method for screening for inhibitors of riboflavin biosynthesis

Info

Publication number: EP1141381A1
Application number: EP99963535A
Authority: EP
Inventors: Adelbert Bacher; Stefan Herz
Original assignee: Individual
Current assignee: Individual
Priority date: 1998-12-15
Filing date: 1999-12-14
Publication date: 2001-10-10
Also published as: JP2002534094A; AU1979500A; WO2000040744A1

Abstract

Methods are described for screening for the presence or absence of inhibition of the activity of enzymes participating in riboflavin biosynthesis. GTP cyclohydrolase II activity is determined through the extent of conversion of GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, catalysed by a protein having a GTP cyclohydrolase II sequence. The activity of 3,4-dihydroxy-2-butanone 4-phosphate synthase activity is detected through the extent of conversion of ribulose 5-phosphate to 3,4-dihydroxy-2-butanone 4-phosphate, catalysed by a protein having a 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence.

Description

Method for screening for inhibitors of riboflavin biosynthesis

The present invention relates to the field of screening for inhibitors of riboflavin biosynthesis. Specifically, the invention relates to methods of screening for inhibitors of GTP cyclohydrolase II or 3,4-dihydroxy-2-butanone 4-phosphate synthase as well as for mutants that exhibit resistance to an inhibitor. The invention also relates to kits of parts for said screening methods. The invention relates further to the application of these screening methods for detecting inhibitors useful as herbicides, or fungicides or as antibacterial agents. The invention relates furthermore to plant enzymes for performing said screening methods.

The human population is situated in an ecology with an ever changing confrontation with dangerous organisms, in the agricultural area weeds, fungi or the like and in the medical or veterinarmedical area pathogenic bacteria, fungi or the like. There is a continued need to develop ever new chemical inhibitors against such undesirable organisms.

In times of a growing world population in conjunction with a growing awareness for the necessity of environmental protection there is a great need for increasing the productivity of agriculturally utilized land. One approach to pursue this problem in the search for novei herbicides or fungicides or the like exhibiting a high effectiveness against all pests such as weeds, fungi or the like, even when applied in small amounts, while not affecting crop plants as well as having little or no toxicity for man or mammals and preferably also all other animals in the relevant ecosystems. In the medical or verterinary medical field there is an ever changing confrontation with pathogens, and notably pathogens with a significant propensity to develop resistance against conventional inhibitors. Inhibitors in this field should be highly effective against a pathogen and applicable in small amounts. They should show little or no toxicity for the patient or animal to be treated. And furthermore, they should be of such a nature that the product of the inhibited metabolic pathway should not be readily transported from the host organism through the cell wall of the pathogen. It is again apparent, that the detection of novel inhibitors satisfying the requirements is a formidable task.

One approach to finding novel inhibitors consists in the modification of known types of inhibitors. This approach promises effects which are not expected to be greatly dissimilar from the effects of the conventional members of these known types of inhibitors.

A more promising approach would be the search for novel types of inhibitors with novel mechanisms for effectiveness. Such a search cannot be guided by a knowledge of the structures of conventional inhibitors. Therefore novel methods of screening libraries of chemical compounds are required for this search.

It is therefore an object to provide a method for screening libraries of chemical test samples for compounds that inhibit with great effectiveness an enzyme in a biochemical pathway that is essential for weed plants, fungi or bacterial pathogens, but not for man or animals and whose inhibitors cannot be compensated by the uptake of the product of the pathway from the enviroment.

We have found that this object can be solved advantageously by a method for screening for inhibitors of riboflavin biosynthesis.

All cellular orgnisms require riboflavin as an indispensible requirement of numerous redox enzymes, many of which are crucial for the metabolism. All plants, fungi and many bacteria generate riboflavin biosynthetically, whereas all animals require a nutritional source of riboflavin (Vitamin B₂) . Therefore, an inhibitor for an enzyme in the biosynthesis of riboflavin in plants, fungi and bacteria would not interfere with the metabolism of animals. Furthermore, the absolute amount of riboflavin for cellular activity is low. Therefore, only small amounts of the enzymes of riboflavin biosynthesis are found in cells. This in turn means that only small amounts of an inhibitor for such an enzyme would be required. This makes the enzymes of riboflavin biosynthesis an ideal target for novel inhibitors.

The biosynthetic pathway of Vitamin B₂ (riboflavin) (Fig. 1 ) has been studied in considerable detail in bacteria and yeasts (for review see Bacher, 1 991 ; Bacher et al., 1 996). The biosynthetic formation of one molecule of riboflavin (7) requires one molecule of GTP and two molecules of ribulose 5-phosphate. GTP (1 ) is initially converted to the committed product, 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5'-phosphate (2) by the enzyme, GTP cyclohydrolase II (step A). This intermediate is converted to 5-amino-6-ribitylamino-2,4( 1 H , 3H)- pyrimidinedione (3) by a sequence of deamination, side chain reduction, and dephosphorylation. The compound (3) is converted to 6, 7-dimethyl-8- ribityllumazine (4) by condensation with 3,4-dihydroxy-2-butanone 4-phosphate (5) which is obtained enzymatically from ribulose 5-phosphate (6) by 3,4- dihydroxy-2-butanone 4-phosphate synthase (step B) . The formation of (5) involves an unusual skeletal rearrangement with a loss of C-4 of the substrate as formate.

The ribA gene of Bacillus subtilis specifies a bifunctional protein catalyzing the formation of the pyrimidine (2) and of the carbohydrate (5) from GTP and ribulose- 5-phosphate, respectively. Similar proteins have been predicted on the basis of DNA sequence information in several other microorganisms such as Bacillus amyloliquefaciens, Synechocystis sp. , Mycobacterium tuberculosis, Actinobacillus pleuropneumoniae or Aquifex aeolicus (Gusarov et a/., 1997; Kaneko et al., 1996; Cole et al. 1998 and 1996, Fuller et al., 1995; Deckert et al., 1998). On the other h a n d , Escherichia coli and th e ye asts Saccharom yces cere visiae , Schizosaccharomyces pompe, Candida guilliermondii, Azospirillum brasilense, have been shown to specify GTP cyclohydrolases II and 3,4-dihydroxy-2-butanone 4- phosphate synthases as separate proteins specified by unlinked genes [Richter et al., 1 992; Oltmans et al., 1 972; Liauta-Teglivets et al. , 1 995; van Bastelaere et al. , 1 995) . The same appears to be true in Haemophilus influenzae, Photobacterium leiognathi, Photobacterium phosphoreum, Archaeoglobus fulgidus, Helicobacter pylori, Dehalospirillum multivorans, Methanococcus jannaschii, Methanobacterium thermoautotrophicum on basis of DNA sequence information {Fleischmann et al., 1 995; Lee et al. , 1 994; Klenk et al. , 1 997; Tomb et al. , 1 997; Bult et al. , 1 996) .

We have discovered that in plants GTP cyclohydrolase II and 3,4-dihydroxy-2- butanone 4-phosphate synthase are fused. This means that the bifunctional enzyme is likely to have compounded structural constraints which make it susceptible for highly specific inhibition and thus a promising target for screening for inhibitors. Such a protein, useful for screening purposes may have both functions combined as in plants or some bacteria; or only one of these functions as in fungi or other bacteria.

It is a first object of the present invention to provide a method for screening chemical test samples for the presence or absence of inhibition of GTP cyclohydrolase II activity for finding an inhibitor.

This first object is achieved by a method for screening for the presence or absence of inhibition of GTP cyclohydrolase II activity, comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a GTP cyclohydrolase II sequence and GTP,

(b) reacting said first mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate, (c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a chemical test sample

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting the level of 2, 5-diamino-6-ribosyIamino-4(3H)-pyrimidinone 5'-phosphate,

(e) determining the presence of inhibition of GTP cyclohydrolase II by observation of whether the level detected in step (d) is lower than the level detected in step (b) .

It is a second object of the invention to provide a method for screening for the presence or absence of resistance to an inhibition of a GTP cyclohydrolase II activity whereby the test sample contains a protein having a mutated plant-type GTP cyclohydrolase II sequence for finding a mutated enzyme that is resistant to a specific inhibitor.

The second object is achieved by a method for screening for the presence or absence of inhibition of GTP cyclohydrolase II comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a mutated plant-type GTP cyclohydrolase II sequence and GTP,

(b) reacting said first mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a specific inhibitor of said GTP cyclohydrolase II,

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting the level of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate,

(e) determining the presence of inhibition or resistance to inhibition respectively, of GTP cyclohydrolase II by observation of whether the level detected in step (d) is similar to the level detected in step (b) . It is a third object of the present invention to provide a method for screening chemical test samples for the presence or absence of inhibition of 3,4-dihydroxy- 2-butanone 4-phosphate synthase activity for finding an inhibitor.

The third object is achieved by a method for screening for the presence or absence of inhibition of 3,4-dihydroxy-2-butanone 4-phosphate synthase activity, comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a 3,4- dihydroxy-2-butanone 4-phophate synthase sequence and ribulose 5- phosphate,

(b) reacting said mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 3,4- dihydroxy-2-butanone 4-phophate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a specific inhibitor for said 3,4-dihydroxy-2- butanone 4-phophate synthase,

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting again the level of 3,4-dihydroxy-2-butanone 4-phophate,

(e) determining the presence of inhibition of 3,4-dihydroxy-2-butanone 4- phophate synthase by observation of whether the level detected in step (d) is lower than the level detected in step (b).

It is a fourth object of the invention to provide a method for screening test samples for the presence or absence of inhibition of 3,4-dihydroxy-2-butanone 4- phosphate synthase activity, whereby the test sample contains a protein having a mutated plant-type 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence, for finding a mutated enzyme that is resistant to a specific inhibitor.

The fourth object is achieved by a method for screening for the presence or absence of inhibition of 3,4-dihydroxy-2-butanone 4-phosphate synthase activity, comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a mutated plant-type 3,4-dihydroxy-2-butanone 4- phosphate synthase sequence and ribulose 5-phosphate,

(b) reacting said mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 3,4- dihydroxy-2-butanone 4-phosphate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a specific inhibitor for said 3,4-dihydroxy-2- butanone 4-phosphate synthase,

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting again the level of 3,4-dihydroxy-2-butanone 4-phosphate,

(e) determining the presence of resistance to inhibition of 3,4-dihydroxy-2- butanone 4-phosphate synthase by observation of whether the level detected in step (d) is similar to the level detected in step (b).

The enzyme sequence may be a plant type for finding herbicides, a fungal type for finding fungicides or a bacterial typ for finding inhibitors against pathogenic bacteria.

For performing the screening methods, kits of parts may be used, whereby the required components are partitioned such that upon mixing of the parts the enzymatic reaction can be initiated at a certain point in time.

Preferably the enzymes are promoted with a bivalent metal ion. In this case the enzymatic reaction may be stopped at a certain point in time by adding a chelating agent for said metal ion.

The level of the products of the reaction of the target enzymes may be determined directly or by chemical or enzymatic derivatization.

The level of the product may either be determined at one predetermined point of time or consecutively at several predetermined points of time.

The invention will now be described in detail.

Sequence determination of plant enzymes

The known amino acid sequence of the RibA protein of B. subtilis [Richter et al., 1993) was used to perform a sequence analysis with Arabidopsis thaliana EST- clone 41G4T7 available from the Arabidopsis Biological Resource Center, Ohio State University, USA, and a significant similarity was found with EST clone 41G4T7. It was determined that this sequence similarity concerned the 3,4- dihydroxy-2-butanone 4-phosphate synthase domain of the RibA protein of B. subtilis. This was surprising since EST 41G4T7 had been previously annotated as riboflavin synthase (Newman etal., 1994).

The entire insert of EST clone 41G4T7 was sequenced by the automated dideoxynucleotide method using a primer walk strategy. The insert (designated i41G4T7) had a length of 2525 bp. The segment comprising bp 109-1297 is similar to the ribA gene of B. subtilis; the segments containing bp 563-1485 and 2262-2505 are identical with the A. thaliana sequence D45165 (Genbank/DDJB/EMBL/GSDB/NCBI accession no.) which was thought to be a GTP cyclohydrolase II (Kobayashi et al., 1995). The transposon IS1 was located at BP 1486-2261.

Sequence comparison with the B. subtilis gene suggested that the EST clone 41G4T7 did not contain the entire 5' part of the gene under study. We therefore performed a Northern blot with mRNA from Arabidopsis thaliana var. Columbia using a hybridization probe obtained by PCR amplification of bp 39-1484 from clone 41 G4T7. This probe contained parts of the putative 3,4-dihydroxy-2- butanone 4-phosphate synthase domain as well as the putative GTP cyclohydrolase II domain. We found a single band of about 2300 bp.

In order to extend the known sequence in the 5' direction, a RACE experiment was performed by reverse PCR using mRNA from A. thaliana as template. The RACE product, which had a length of 800 bp, was sequenced and was shown to contain 556 bp which were not present in clone 41 G4T7. The coding region of the extended cDNA contains 1 629 bp specifying a predicted protein with 543 aa and a mass of 59,055 Da. The cognate gene is designated ribA . The sequence is shown in Annex B. The 5' untranslated region is 284 bp long. The 3' untranslated region has a length of 372 bp.

The putative 3,4-dihydroxy-2-butanone 4-phosphate synthase domain of the Arabidopsis gene is preceded by a sequence of about 1 20 amino acids which is devoid of similarity to any sequence in the database. The first 25 amino acids of the protein contain 1 3 serine residues (including a cluster of 4 consecutive serine residues). Thirtyfour residues in the N-terminal 1 20 amino acid residues are serine or threonine.

The chromosomal ribA gene of Arabidopsis was amplified in two parts by PCR using chromosomal DNA as template. The amplificates were sequenced. It was found that this gene (EMBL database accession no. AJ000053) includes six introns with a total of 700 bp.

Complementation experiments

The coding region of the A. thaliana gene was inserted into the expression vector pNCO 1 1 3 (Stuber et al. , 1 990) yielding the plasmid pAE. This plasmid was electrotransformed into ribA and ribB mutant strains of E. coli which are deficient of 3,4-dihydroxy-2-butanone 4-phosphate synthase and GTP cyclohydrolase II, respectively, and which are, therefore, unable to grow on LB medium without supplementation of riboflavin (Katzenmeier, 1 991 ). The plasmid pNCO 1 1 3 transformed into the mutants served as a negative control. The recombinant mutant strains carrying the plasmid with the ribA gene of Arabidopsis grow at normal rate in the absence of external riboflavin. It follows that the ribA gene of Arabidopsis directs the synthesis of a protein which can serve as GTP cyclohydrolase II and as 3,4-dihydroxy-2-butanone 4-phosphate synthase in £. coli cells. The A. thaliana gene therefore codes for a bifunctional GTP cyclohydrolase 11/ 3,4-dihydroxy-2-butanone 4-phosphate synthase.

Cloning of the ribA gene of tomato

A gene with similartiy to the ribA gene of A. thaliana was amplified from a tomato cDNA library by PCR using degenerate primers. The DNA segment was sequenced using a primer walk strategy and was shown to contain an open reading frame (EMBL database accession no. AJ002298) of almost the same size as the A. thaliana gene (Annex C) . The predicted protein sequence contains 552 amino acids and has a calculated mass of 59793 Da. The predicted protein is similar to the RibA protein of A. thaliana. Most notably, it also has a serine and threonine rich N-terminus of about 1 20 amino acids preceding the 3,4-dihydroxy-2-butanone 4-phosphate synthase domain. Apart from the high serine/threonine content, the N-terminus of the tomato gene has little similarity to the A. thaliana N-terminus. The following 3,4-dihydroxy-2-butanone 4-phosphate synthase and cyclohydrolase II domains are very similar to the Arabidopsis enzyme.

By the method here described the corresponding gene and protein sequence of any other organism can be determined.

The deduced amino acid sequence of the ribA gene of A. thaliana shows also similiarity with bifunctional GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4- phosphate synthase sequences from Actinobacillus pleuropneumoniae and with putative bifunctional enzymes from Bacillus amyloliquef aciens , M. tuberculosis and Synechocystis sp. The A. thaliana gene is also similar to genes from Photobacterium phosphoreum and Photobacterium leiognathi. The N-terminal 3,4- dihydroxy-2-butanone 4-phosphate synthase domain of the A. thaliana enzyme shows similarity to monofunctional 3,4-dihydroxy-2-butanone 4-phosphate synthase sequences not only from Escherichia coli, but also H. influenzae and S. cerevisiae. The C-terminal GTP cyclohydrolase II domain shows similarity to monofunctional GTP cyclohydrolase II sequences from not only E. coli, but also H. influenzae, S. cerevisiae, Candida guilliermondii, P. phosphoreum and Azospirillum brasilense.

Expression and purification of GTP cyclohydrolase II/3,4-dihydroxy-2-butanone 4- phosphate synthase from plants

Originator for the construction of an expression vector is cDNA of a plant, preferably of a dicotyle or monocotyle plant, for example cDNA from Arabidopsis thaliana or Lycopersicon esculentum. The ribA gene is amplified by PCR with specific primers and cDNA from the corresponding plant as template. In a variant process the gene may be amplified in two overlapping parts. The two parts are digested with a restriction enzyme in the overlapping area and ligated together afterwards to yield the complete gene. Alternatively the cDNA of one or both parts may originate from an existing EST-clone. If only the GTP cyclohydrolase II part or the 3,4-dihydroxy-2-butanone 4-phosphate synthase part of ribA should be expressed, only the corresponding part of the gene is amplified with the above described methods.

The RibA protein from various plants for example A. thaliana or L. esculentum includes a signal sequence of about 1 20 amino acids which was found not to be essential for enzyme activity. This signal sequence may be excluded in the recombinant DNA construct. The amplified DNA fragment is modified by two consecutive PCR amplifications with modifying primers. In the first PCR reaction a ribosomal binding site preceding the start codon at an optimal distance is introduced at the 5'-end. A recognition site for a restriction enzyme, for example Sail, BspMI or Pvu1 is introduced at the 3'-end. The preferred recognition site is Sail. In the second PCR reaction the product of the first PCR reaction is used as template. At the 5'-end a recognition site for the restriction enzyme EcoRI preceding the ribosomal binding site is introduced with a modifying primer. The amplified DNA fragment is digested with the corresponding restriction enzymes, for example EcoRI and Sail. This DNA fragment is inserted into a vector DNA capable of autonomous replication in the host microorganism to give a recombinant plasmid containing said DNA (Fig. 2) . This recombinant plasmid is used to transform the host microorganism, for example Escherichia coli or Bacillus subtilis. The preferred host is E. coli. The expression of plant proteins may be poor in the host organism. To enhance the expression level and/or to simplify the purification of the protein, the recombinant plasmid may include a gene or a part of a gene without stop codon preceding the ribA gene or parts of it in the same reading frame. A preferred gene for this purpose is the malE gene from E. coli. The expression of such a recombinant DNA generates a fusion protein between the maltose binding protein (MBP) from E. coli and the plant RibA protein. Various constructs are shown in Fig. 3. A construct with a signal sequence S is shown in Fig. 3a, with a maltose binding sequence malE and a signal sequence S in Fig. 3b and with a maltose binding sequence malE in Fig. 3c.

The strains harbouring the expression vectors can be cultivated in conventional culture media at 1 5 to 40 °C. The preferred culture medium is Luria Bertani medium and the preferred temperature is 37 °C. The E. coli strains are induced with 0.2 to 5 mM isopropyl-R-D-thiogalactosid at an optical density from 0.5 to 0.8. The cells are incubated between 2 and 1 2 h, preferably 5 h. The cells are harvested by centrifugation and washed with saline. The cells are lysed with lysozyme and/or disrupted with a sonifier. The MBP-ribA fusion protein is purified from crude extract by affinity chromatography with an amylose resin.

Screening for the presence or absence of inhibition of GTP cyclohydrolase II activity

GTP is converted to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate by the action of GTP cyclohydrolase II. The enzyme can be promoted by a bivalent metal ion such as Ba^{2 +}, Ca^{2 +}, Sr^{2 +}, Co^{2 +} , Fe^{2 +}, Mg^{2 +}, Mn^{2 +} or Zn^{2 +} . The preferred ion is Mg^{2 +} . The reaction mixture should preferably contain an antioxidative substance such as dithiothreitol (DTT), dithioerythritol (DTE), butylhydroxyanisole (BHA) or butylhydroxytoluene (BHT) . The assay can be started by adding one of the needed substances to a mixture of the others. Preferably the enzyme is added to a solution of GTP, Mg²⁺ , DTT in a buffer at pH 6 to 9,5, preferably 8, 5. The reaction can be incubated for 1 to 60 min at 10 to 40 °C. Preferable it is incubated for 20 min. at 37 °C. The assay can be stopped by denaturating the enzyme with trichloroacetic acid, acetone, sodium dodecyl sulfate or heating at temperatures between 60 to 100 °C. The assay can also be stopped by chelating the metal ion with complexing agent such as EDTA, iminodiacetic acid, 8-hydroxyquinolin, diphenylcarbazid, dithizon or glyoxal-bis-(2- hydroxyanil) . The preferred complexing agent is EDTA.

The assay is carried out with otherwise identical mixtures with and without test sample of a possible inhibitor. The enzyme product 2,5-diamino-6-ribosylamino- 4(3H)-pyrimidinone 5'-phosphate can be detected directly without derivatization, preferably photometrically, preferentially after purification by HPLC. The pyrimidinone can be identified by UV absorbance at 293 nm. The extinction coefficient is 1 2, 100 M^"1cm^"1. The product can also be monitored by diode array multiwavelength detection (200 - 600 nm). The assay may also be performed in a quartz cuvette without stopping the reaction. The reaction rate can then be determined directly by monitoring the absorbance of GTP at 252 nm and of 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate at 293 nm. The pyrimidinone can also be detected by chemical derivatization, preferably by reaction with a vicinal dioxo compound (Fig. 4) . Preferable are vicinal diketo compounds of the formula R¹-CO-CO-R², wherein R¹ and R² are independently aliphatic, alicyclic, aromatic or heteroaromatic residues, such as diphenyldiketone, phenyl-methyl-diketone, camphoro quinone; preferably C,_₆ alkyl rests. The most preferred diketone is diacetyl (2,3-butanedione) (8). The reaction with 2,5-diamino- 6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate yields 6,7-dimethylpterin (9), which can be detected fluorometrically at an absorbance wavelength at 365 nm and an emission wavelength at 435 nm.

The enzyme activity of GTP cyclohydrolase II can also be monitored after enzymatic derivatization, preferably by converting 2, 5-diamino-6-ribosylamino- 4(3H)-pyrimidinone δ'-phosphate into 5-amino-6-ribitylamino-2,4( 1 H, 3H)- pyrimidinedione 5'-phosphate ( 10) by action of a fungal, preferably yeast, 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase (Fig. 4); for the sequence Annex D is pointed out. This reductase can be expressed in E. coli with additional N- or C-terminal histidines by standard molecular biological techniques with DNA from Saccharomyces cerevisiae as template. Before used in the assay the reductase has to be purified for example on a metal chelate affinity column. The conversion needs NADPH or NADH as cosubstrate. The amount of 2,5- diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate can be calculated by the consumption of NADH which can be monitored photometrically at 340 nm.

The screening can be done with a bifunctional enzyme of a monocotyledonous or dicotyledonous plant. It is also possible to use only the GTP cyclohydrolase II domain in a first step screening and bifunctional enzyme in a second step screening. It is also possible to use a bacterial or fungal enzyme. Screening for the presence or absence of inhibition of 3,4-dihydroxy-2-butanone 4-phosphate synthase activity

Ribulose-5-phosphate is converted to 3,4-dihydroxy-2-butanone 4-phosphate by the action of 3,4-dihydroxy-2-butanone 4-phosphate synthase. The enzyme requires a bivalent metal ion such as Ba^{2 +} , Ca^{2 +}, Sr^{2 +} , Co^{2 +}, Fe^{2 +} , Mg^{2 +}, Mn^{2 +} or Zn^{2 +} . The preferred ion is Mg^{2 +} . The assay can be started by adding one of the needed substances to a mixture of the others. Preferably the enzyme is added to a solution of ribulose-5-phosphate and Mg^{2 +} in buffer at pH 6 to 9, 5, preferably 7,5. The assay is carried out with otherwise identical mixtures with and without a test sample of a possible inhibitor. The reaction can be incubated for 1 to 90 min at 10 to 40 °C. Preferably it is incubated for 60 min. at 37 °C. The substrate for 3,4-dihydroxy-2-butanone 4-phosphate synthase, ribulose 5-phosphate is commercially available, but at considerable cost. It is preferably generated in situ by isomerization of ribose 5-phosphate catalyzed by pentose phosphate isomerase. In this case a mixture containing preferably pentose phosphate isomerase, ribose- 5-phosphate and Mg^{2 +} in buffer at pH 6,0 to 9,5 incubated for 1 to 20 min before adding 3,4-dihydroxy-2-butanone 4-phosphate synthase and optionally the test sample. The assay can be stopped by chelating the metal ion with complexing agent such as EDTA, iminodiacetic acid, 8-hydroxyquinolin, diphenylcarbazid, dithizon or glyoxal-2-bis-(2-hydroxyanil). The preferred complexing agent is EDTA. For detection enzymatic derivatization is possible. Preferably, the product 3,4- dihydroxy-2-butanone 4-phosphate can be converted enzymatically with 5-amino- 6-ribitylamino-2,4( 1 H,3H)-pyrimidinedione to 6,7-dimethyI-8-ribityllumazine in the presence of 6,7-dimethyl-8-ribitylIumazine synthase or to riboflavin in the presence of 6,7-dimethyl-8-ribityl-lumazine synthase and riboflavin synthase. 5-Amino-6- ribitylamino-2,4( 1 H,3H)-pyrimidinedione decomposes rapidly in the presence of molecular oxygen and has to be stored, therefore, as aqueous solution containing an antioxidative substance such as dithiothreitol (DTT) or dithioerythrol (DTE) at temperatures below 0 °C. The assay can be stopped and the detection reaction started by adding a solution of complexing agent, 5-amino-6-ribitylamino- 2,4( 1 H , 3H)-pyrimidinedione, antioxidative substance, 6 , 7-dimethyl-8- ribityllumazine synthase and optionally riboflavin synthase (Fig. 5) . After incubation for 10 to 60 min at 20 to 40 °C the detection reaction can be stopped by denaturating the enzymes with trichloroacetic acid, acetone, sodium dodecyl sulfate or heating at temperatures between 60 to 100 °C. 6,7-Dimethyl-8- ribityllumazine can be detected photometrically at 41 0 nm, if only the lumazine synthase is added. Riboflavin can be detected photometrically at 445 nm, if the lumazine synthase and the riboflavin synthase are added . The extinction coefficient for riboflavin (pH 1 , 445 nm) is 1 1 ,500 M^"1cm^"1. The extinction coefficient for 6,7-dimethyl-8-ribityllumazine (pH 1 , 41 0 nm) is 1 0,300 M^cm^"1. There are two molecules of 3,4-dihydroxy-2-butanone 4-phosphate required for the formation of one molecule riboflavin.

3,4-Dihydroxy-2-butanone 4-phosphate can also be detected after chemical derivatization, preferably with an aromatic or heteroaromatic ortho-diamine (Fig. 5) . The aromatic or heteroaromatic ring may be substituted or unsubstituted. It may contain 1 to 3 substituents selected for the group of alkyl, halogen, alkoxy.

It is possible to use the bifunctional enzyme of a monocotyledonous or dicotyledonous plant. It is also possible to use only the 3,4-dihydroxy-2-butanone 4-phosphate synthase domain in a first step screening and bifunctional enzyme in a second step screening. It is also possible to use a bacterial or fungal enzyme.

The screening methods described above may readily be used for screening for inhibition resistance after corresponding modification. It is merely necessary to add a specific inhibitor (previously determined by screening) and to use a biological test sample which contains a mutant of the plant-type enzyme in question.

The invention will now be described by specific Examples.

Reference Example 1 Isolation of the A. thaliana ribA gene

1 g of 2 weeks old Arabidopsis thaliana var. Columbia plants (stems and leafs) were frozen and homogenised in liquid nitrogen. 8 ml of 50 mM trisodium citrate containing 600 g guanidine thiocyanate, 5 g sodium-N-lauroylsarcosine, and 5 ml 2-mercaptoethanol per liter were added. This mixture was added carefullly to 3 ml of a sterile solution containing 959 g CsCI and 37.2 g EDTA per liter. The mixture was centrifuged at 33,000 rpm at 1 8°C for 24 h. The supernatant was discarded and the pellet was airdried for 10 min. The pellet was dissolved in 360 μl H₂0 (bidestillated, sterile) . The solution was centrifuged at 1 4,000 rpm for 1 0 min. The supernatant was mixed with 40 μ\ of 3 M sodium acetate and 1 ml of ethanol. The RNA was precipitated overnight at -20°C, centrifuged at 1 4,000 rpm at 4°C for 1 5 min and washed twice with 500 μl of 75 % ethanol. The pellet was airdried and dissolved in 500 μl of H₂0 (bidestilled, sterile) . mRNA from this crude RNA fraction was isolated with the Oligotex mRNA-lsolation kit (Quiagen). The RNA solution (500 μl) was mixed with 500 μl 20 mM Tris hydrochloride, pH 7.5 containing 1 M NaCl, 2 mM EDTA and 0.2 % SDS. 30 μl Oligotex suspension were added and the mixture was incubated for 3 min at 65 ° and for 1 0 min at room temperature. The suspension was centrifuged for 2 min at 14,000 rpm and the supernatant was aspirated. The pellet was washed twice with 1 ml of 10 mM Tris hydrochloride, pH 7.5 containing 1 50 mM NaCl and 1 mM EDTA. The mRNA was eluted twice with 50 μl of preheated (70°C) 5 mM Tris hydrochloride buffer, pH 7.5

A. thaliana mRNA (4 μg) was electrophoresed at 40 V on a 2.2 M formaldehyde 1 % agarose gel. 4 μl of a RNA ladder (Gibco) was electrophoresed as a reference. The reference lane was cut off and stained in a 0.1 % toluidine blue solution for 1 0 min The other part of the gel was washed 4 times with H₂O (DEPC treated) and transferred overnight to a Nytrans nylon membrane with 0.3 M trisodium citrate, pH 7.0 containing 3 M NaCl using the turboblotter system from Schleicher and Schuell. The probe was amplified from plasmid 41 G4T7 by PCR. The plasmid was isolated from 5 ml of fresh overnight culture of EST-clone 41 G4T7 using the mini plasmid isolation kit from Quiagen. The bacterial pellet was resuspended in 0.3 ml of 50 mM Tris hydrochloride, pH 8.0 containing 10 mM EDTA and 100 μg/ml RNase. 0.3 ml of 200 mM sodium hydroxide containing 1 % SDS were added and incubated 5 min at room temperature. 0.3 ml of chilled 3.0 M sodium acetate pH 5.5 were added and incubated on ice for 1 0 min. The mixture was centrifuged for 1 5 min at 14,000 rpm in a minifuge. The supernatant was removed and applied onto a Quiagen-tip 20 which was previously equilibrated with 1 ml of 50 mM MOPS, pH 7.0 containing 750 mM NaCl, 1 5 % ethanol and 0.1 5 % Triton X-1 00. The Quiagen-tip was washed 4 times with 1 ml of 50 mM MOPS, pH 7.0 containing 1 000 mM NaCl and 1 5 % ethanol. The DNA was eluted with 0.8 ml of 50 mM Tris hydrochloride, pH 8.5 containing 1 250 mM NaCl and 1 5 % ethanol. The DNA was precipitated with 0.7 volumes of isopropanol, centrifuged at 14,000 rpm for 30 min and washed with 1 ml of cold 70 % ethanol. The DNA sequence of 41 G4T7 was previously determined with a primer walk strategy. Sequencing was performed by the automated dideoxynucleotide method using an ABI Prism 377 DNA sequencer from Applied Biosystems Inc. with the ABI Prism Sequencing Analysis Software. EST-clone 41 G4T7 was obtained from the Arabidopsis Biological Resource Center, Ohio State University, USA. The PCR mixture contained 25 pmol primer CTCCTCCTGCACCAGCCAATGG, 25 pmol primer TCAAGTTTCTCAGACAG ATCAAATG, 2U of Taq polymerase, 10 μl of buffer (Primezyme, Biometra), 1 .6 ng of plasmid 41 G4T7, and 20 nmol dNTPs in a total volume of 100 μl H₂0.

The mixture was denaturated at 94°C for 5 min. Then 25 cycles (30 sec at 94°C, 45 sec at 50°C, 90 sec at 72°C) were performed. After incubation for 7 min at 72°C, the mixture was cooled at 4°C and the DNA was purified with a PCR purification kit (Quiagen) . 5 Volumes of buffer PB (Quiagen) were added to 1 volume of the PCR reaction, applied to a Quiaquick column and centrifuged for 1 min at 1 4,000 rpm. The flow through was discarded. 0.75 ml buffer PE (Quiagen) were added to the column and centrifuged as before. The flow through was discarded and the column was centrifuged for an additional 1 min at 14,000 rpm. The column was placed in a clean 1 .5 ml eppendorf tube. 50 μl of H₂0 (bidestilled, sterile) were added to the column and it was centrifuged for 1 min at 14,000 rpm. The flow through contains the purified DNA. 105 ng of the purified DNA in 1 5 μl H₂0 were heated at 1 00°C for 5 min. After cooling on ice, 5 μl of buffer containing random hexamer primers, dATP, dGTP, dTTP, 5 μl of ³²P-dCTP (50μCi) and 0.8 μl of Klenow polymerase (4 U) were added. The mixture was incubated for 1 h. The probe DNA (bp 39-1484 from EST sequence 41 G4T7) was precipitated two times with 2 M NH₄SO₄. The pellet was dissolved in 200 μl of H₂0.

The membrane was prehybridized in a mixture of 50 % formamide, 50 % SSPE/Denhards solution/SDS ( 1 .0 g Ficoll, 1 .0 g polyvinylpyrrolidone, 1 .0 g BSA, 87.6 g NaCl, 1 3.8 g NaH₂PO₄, 3.7 g EDTA, 10 g SDS per liter) for 1 h at 42°C. 200 μl probe were added, and hybridization was performed at 42°C in 50 % formamide, 50 % SSPE/Denhards solution/SDS for 1 2 h. The membranes were then washed twice with 2xSSPE containing 0.1 % SDS at room temperature for 1 5 min. The radioactive bands were detected for 3.5 h on a Photolmager (Molecular Dynamics) . A single band of about 2300 bp was found.

A 5' RACE was performed with the 5' RACE system from GibcoBRL. The cDNA was generated with a specific primer from A. thaliana RNA. The mixture contained 2.5 pmol of primer TCAACAGATGCTTCAGTGTGTCC and 990 ng of A. thaliana RNA in a total volume of 1 5 μl. The mixture was denaturated at 70°C for 10 min and cooled on ice. 2.5 μl of 10x reaction buffer (Gibco), 3 μl 25 mM MgCI₂, 1 μl 10 mM dNTPs and 2.5 μl 0.1 M dithiothreitol were added. The mixture was incubated at 42°C for 2 min, 1 μl (200 U/μl) of reverse transcriptase Superscriptll (Gibco) were added. The mixture was incubated at 65 °C for another 1 5 min. It was then centrifuged, the temperature was adjusted to 55°C, and 1 μl of RNAseH (2 U/μl) was added. After another 10 min incubation at 55 °C the mixture was cooled on ice.

Subsequently the cDNA was amplified using an anchor primer (GibcoBRL) and a specific nested primer (CCTTCATTTTCCCTATCTTCATCATC) . The product was purified by electrophoresis in a 2 % agarose gel. The band at 800 bp was extracted using a gel extraction kit (Quiagen) and sequenced. The DNA fragment was excised from the agarose gel with a scalpel. 3 volumes of buffer QX1 (Quiagen) were added to 1 volume of the excised gel and incubated at 50°C for 1 0 min. One gel volume of isopropanol was added. To bind DNA, the sample was applied to a Quiaquick column and centrifuged for 1 min at 14,000 rpm. The flow through was discarded. 0.75 ml buffer PE (Quiagen) were added to the column and centrifuged as before. The flow through was discarded and the column was centrifuged for an additional 1 min at 14,000 rpm. The column was placed in a clean 1 .5 ml eppendorf tube. 50 μl of H₂0 (bidestilled. sterile) were added to the column and it was centrifuged for 1 min at 1 4,000 rpm. The flow through contained 4.2 μg of the purified DNA.

Reference Example 2

Construction of an expression clone

Using plasmid from EST clone 41 G4T7 as template, the cDNA insert was amplified from bp -75 to 1 485 by PCR. The reaction mixtures contained 1 0 pmol of primer TCAAGTTTCTCAGACAGATCAAATG, 1 0 pmol primer GAAACAGCT ATGACCATGATTACG, 4.5 ng of plasmid 41 G4T7, 2U of Taq polymerase, 1 0 μl of buffer (Primezyme, Biometra), and 20 nmol of dNTPs in a total volume of 100 μl.

The mixture was denaturated at 94°C for 5 min. 25 PCR cycles (60 sec at 94°C, 60 sec at 50°C, 1 20 sec at 72°C) were performed. After another 7 min incubation at 72°C, the mixture was cooled at 4°C, and the DNA was purified with a PCR purification kit (Quiagen). The PCR product and the RACE product from (Reference Example 1 ) were digested with restriction enzyme Bsgl (New England Biolabs) . 5 μl New England Biolabs buffer 4, 1 μl S-adenosylmethionin (4 mM), 5 μl Bsgl (7.5 U), 40 μl PCR product ( 1 .6 μg) resp. 40 μl RACE product (3.2 μg).

Both fragments were incubated at 37 °C for 2 h, purified as before and ligated together with T4-ligase, 4 μl of 5x buffer (Gibco), 1 μl of T4-ligase (4U), 2 μl of PCR product (60 fmol), 0.5 μl of RACE product (65 fmol), and 1 2.5 μl of H₂0.

The mixture was incubated at 4°C for 1 2 h. The completed gene was amplified by PCR with specific terminal primers. At the 5' end a recognition site for the restriction enzyme EcoRI preceding a ribosomal binding site at an optimal distance to the start codon was introduced by PCR with modifying primers. At the 3' end a recognition site for the restriction enzyme Sail was introduced after the stop codon by PCR with modifying primer.

First PCR

1 0 pmol primer GAGGAGAAATTAACTATGTCTTCCATCAATTTATCCTC, 1 0 pmol primer ACGCGTCGACGGTTCGTCCTGGTTTTTAAGC, 2U of Taq polymerase, 1 0 μl of buffer (Primezyme, Biometra), 1 μl of the ligation mixture, and 20 nmol of dNTPs in a total volume of 100 μl.

The mixture was denaturated at 94°C for 5 min. Then 25 cycles (60 sec at 94°C, 60 sec at 50°C, 1 20 sec at 72°C) were performed. After another 7 min incubation at 72°C, the mixture was cooled at 4°C and the DNA was electrophoresed on a 0.8 % agarose gel. The band at 1 650 bp was purified with a gel extraction kit (Quiagen).

Second PCR (Two identical PCRs with 100 μl each were performed to obtain a higher yield) 10 pmol primer CAATTTGAATTCATTAAAGAGGAGAAATTAACTATG, 1 0 pmol primer ACGCGTCGACGGTTCGTCCTGGTTTTTAAGC, 2U of Taq polymerase, 1 0 μl of buffer (Primezyme, Biometra) , 4 μl of purified PCR1 product, and 20 nmol dNTPs in a total volume of 100 μl.

The mixture was denaturated at 94°C for 5 min. Then 25 cycles (60 sec at 94°C, 60 sec at 50°C, 1 20 sec at 72°C) were performed. After another 7 min incubation at 72°C the mixture was cooled at 4°C and the DNA was purified with a PCR purification kit (Quiagen) . Plasmid pNCO1 1 3 (Stϋber et al., 1 990) was isolated as described for plasmid 41 G4T7 in (Reference Example 1 ) .

The PCR product and the plasmid pNCO 1 1 3 were digested with the restriction enzyme Sail ( 1 0 μl Sail buffer (NEB), 1 μl of BSA 1 00 μg/ml, 40 U of Sail (NEB), 2.4 μg of PCR2 product resp. 4.2 μg of pNC01 1 3 in a total of 1 00 μl) at 37°C for 3 h, purified with a PCR purification kit (Quiagen) and digested with the restriction enzyme EcoRI (20 μl of OPA buffer (Phamacia), 48 μl of Sail digested PCR2 product resp. 48 μl of Sail digested pNCO 1 1 3, 2 μl (20 U/μl) of EcoRI (Phamacia) and 30 μl of H₂0) at 37 °C for another 2 h and purified with a PCR purification kit. The digested PCR2 product and plasmid pNCO 1 1 3 were ligated together with T4-ligase, yielding plasmid pAE: 44 fmol of pNCO1 1 3, 1 1 1 fmol of PCR2 product, 4 μl of buffer (Gibco), and 1 μl of T4 ligase ( 1 U) in a total of 20 μl.

The mixture was incubated over night at 4°C, purified with a PCR purification kit and transformed into electrocompetent £. coli XL I cells by electroporation.

Preparation of the electrocompetent cells: One liter of Luria-Bertani-media was inoculated with 1 /1 00 volume of fresh overnight culture. The cells were grown at 37 °C with vigorous shaking to an optical density of 0.5 to 0.7. The cells were chilled on ice for 20 min and centrifuged in a cold rotor at 4,000 g for 1 5 min at 4 °C. The supernatant was removed and the pellet resuspended in 1 liter of ice-cold sterile 10 % glycerol. The cells were centrifuged two times as described before and resuspended the first time in 0.5 liter and the second time in 20 ml of ice-cold sterile 1 0 % glycerol. The cells were centrifuged an additional time and the pellet was resuspended to a final volume of 2 to 3 ml in ice-cold 10 % glycerol. This suspension was frozen in aliquots of 80 μl and stored in liquid nitrogen.

Electro-transformation using the Gene Pulser apparatus from Biorad: The electrocompetent cells were thawn at room temperature and placed on ice. 40 μl of the cell suspension were mixed with 1 μl of the ligation mixture and transferred into a sterile 0.2 cm cuvette (Biorad). The suspension was shaked to the bottom and the cuvette was placed into the chamber slide. The chamber slide was pushed into the chamber and a pulse was applied (2.50 kV, 25 μF, Pulse Controller 200 Ω). The cuvette was removed from the chamber and the cells were suspended in 1 ml of SOC medium (2 % casein hydrolysate, 0.5 % yeast extract, 1 0 mM NaCl, 2.5 mM KCl, 10 mM MgCI₂, 1 0 mM MgSO₄, 20 mM glucose) . The suspension was shaked 1 h at 37 °C and plated on LB media supplemented with 1 50 mg ampicillin per liter. Plasmids from different clones were isolated (pAE 1 -pAE1 1 ) as described in Reference Example 1 for 41 G4T7.

Reference Example 3

Construction of a malE-ribA fusion clone

The ribA gene was amplified by PCR using plasmid pAE 1 1 (from A. thaliana ribA expression clone) as template. Plasmid pAE1 1 was isolated as described for plasmid 41 G4T7 in (Reference Example 1 ). A recognition site for the restriction enzyme EcoRI preceding the start codon was introduced at the 5' end with a modifying primer.

PCR mixture: 1 0 pmol primer GTTCAGAATTCATGTCTTCCATCAATTT ATCCTC, 10 pmol primer ACGCGTCGACGGTTCGTCCTGGTTTTTAAGC, 2U Taq polymerase, 1 0 μl buffer (Primezyme, Biometra), 1 .7 ng plasmid pAE1 1 , and 20 nmol dNTPs in a total volume of 100 μl.

The mixture was denaturated at 94 °C for 5 min. Then 25 cycles (60 sec at 94 °C, 60 sec at 50 °C, 1 20 sec at 72°C) were performed. After another 7 min incubation at 72°C the mixture was cooled at 4°C and the DNA was purified with a PCR purification kit (Quiagen).

The PCR product and the plasmid pMal-c2 (New England Biolabs) were digested with the restriction enzyme Sail ( 10 μl Sail buffer (NEB) , 1 μl BSA 100 μg/ml, 60 U Sail (NEB) , 2.0 μg PCR product resp. 0.4 μg pMal-c2 in a total volume of 100 μl) at 37 °C for 2 h, purified with a PCR purification kit (Quiagen) and digested with the restriction enzyme EcoRI (20 μl OPA buffer (Phamacia), 48 μl Sail digested PCR product resp. 48 μl Sail digested pMal-c2, 3 μl EcoRI (20 U/μl) (Phamacia) and 30 μl H₂0) at 37 °C for another 2 h and purified with a PCR purification kit. The digested PCR product and plasmid pMal-c2 were ligated together with T4-ligase, yielding plasmid pMal_ribA: 9 fmol pMal-c2, 30 fmol PCR product, 4 μl buffer (Gibco), and 1 μl T4-ligase ( 1 U) in a total volume of 10 μl. The mixture was incubated over night at 4°C. This plasmid contains the A. thaliana ribA gene in the same reading frame as the malE gene, resulting in the expression of a MBP-RibA fusion protein after induction with IPTG. The plasmid pMal ribA was transformed into E. coli XL I cells by electroporation as described in (Reference Example 2) .

Reference Example 4

Construction of a malE-ribA fusion clone without transit peptide sequence

The ribA gene exclusive the first 384 bp coding for a putative signal sequence was amplified by PCR using plasmid pAE1 1 (from A. thaliana ribA expression clone) as template. A recognition site for the restriction enzyme EcoRI preceding serine1 28 was introduced at the 5' end with a modifying primer. PCR mixture: 10 pmol primer GTTCAGAATTCTCTTCTATCCCCGAGGC, 10 pmol primer ACGCGTCGACGGTTCGTCCTGGTTTTTAAGC, 2 U Taq polymerase, 1 0 μl buffer (Primezyme, Biometra), 1 .7 ng plasmid pAE 1 1 , and 20 nmol dNTPs in a total volume of 1 00 μl.

The mixture was denaturated at 94 °C for 5 min. Then 95 cycles (60 sec at 94°C, 60 sec at 50°C, 1 20 sec at 72°C) were performed. After another 7 min incubation at 72°C the mixture was cooled at 4°C and the DNA was purified with a PCR purification kit (Quiagen).

The further steps were analogous to the construction of pMal_ribA in Reference Example 3. The plasmid encoding a MBP-RibA fusion protein without the putative transit peptide sequence of RibA was named pMal_ribAS.

Preparation Example 1

Preparation and purification of the MBP-RibA fusion proteins

0.5 I Luria Bertani (LB) medium containing 75 mg ampicillin were inoculated with 20 ml overnight culture of E. coli strain XL I harboring plasmid pMal ribA resp. pMal_RibAS. The culture was grown in shaking culture at 37 °C. At an optical density (600 nm) of 0.8 the culture was induced with 1 mMol IPTG. The culture was grown for another 5 h. The cells were harvested by centrifugation for 20 min at 5000 rpm and 4 °C. The cells were washed with 0.9 % NaCl solution, centrifugated as above and frozen at -20 °C for storage.

The cells were thawed in 1 0 ml 20 mM Tris hydrochloride pH 7.4 containing 200 mM NaCl, 1 mM EDTA, 6 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol and 1 mg lysozyme (buffer A). The mixture was incubated at 37°C for 1 h, cooled on ice and sonified 6 x 10 sec (Branson sonifier level 4). The suspension was centrifugated at 1 5,000 rpm at 4 °C for 20 min. The supernatant was diluted 1 : 5 with buffer A and applied to a 1 ml column of amylose resin (New England Biolabs) previously equilibrated with 8 ml buffer A. The column was washed with 1 2 ml buffer A without lysozyme. The fusion protein was eluted from the column with 4 ml 20 mM Tris hydrochloride pH 7.4 containing 200 mM NaCl, 1 mM EDTA and 10 mM maltose. 9.8 mg MBP-ribAS and 5.6 mg MBP-ribA protein were obtained. The proteins were identified by SDS-PAGE. MBP-ribAS showed a band at 89 kDa and MBP-ribA showed a band at 102 kDa.

Preparation Example 2 "reductase"

Construction and purification of a 2,5-diamino-6-ribosylamino-4 (3H)- pyrimidinone-5-phosphate reductase expression clone.

A PCR was carried out containing 1 0 pmol primer CATGCCATGGGTTCTTTGAC ACCACTGTGTGAAG, 10 pmol primer TATTATGGATCCTTAGTCATCGGCCAG TCTCGC, 2 U of taq polymerase, 1 0 μl of buffer (Primezyme, Biometra), 20 nmol of dNTPs and 30 ng of Saccharomyces cerevisiae DNA in a total of 1 00 μl. The mixture was denaturated at 94°C for 5 min. Then 30 cycles (30 sec at 94°C, 30 sec at 50°C, 90 sec at 72°C) were performed. After another 7 min. incubation at 72°C the mixture was cooled at 4°C and the DNA was purified with a PCR purification kit (Quiagen). The PCR product and the plasmid pNCO-H6 were digested with the restriction enzymes Ncol and BamHI (20 μl OPA buffer (Pharmacia), 40 U BamHI, 40 U Ncol and 2.5 μg PCR product resp. 1 μg pNCO-H6 in a total of 100 μl) at 37°C for 3 h. The digested DNA was purified with a PCR purification kit (Quiagen) and ligated together with T4-ligase (40 fmol of pNCO-H6, 80 fmol of the PCR product, 4 μl of buffer (Gibco), 1 U T4-iigase in a total of 20 μl). The mixture was incubated overnight, purified with a PCR purification kit (Quiagen) and transformed in electrocompetent E. coli XL1 cells as described in Reference Example 1.

Plasmid pNCO-H6 is essentially identical to plasmid pNCO1 13 (see reference Example 2) but the DNA sequence between the Ncol and the BamHI recognition site is substituted by the DNA sequence: CATGCACCACCACCACCACCACGC GTCCATGGCCGCGGATCC .

0.5 I Luria Bertani (LB) medium containing 75 mg ampicillin were inoculated with 20 ml overnight culture of E. coli cells harbouring the plasmid pNCO-H6 with the gene for 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone-5-phosphate reductase. The culture was grown in a shaking culture at 37 °C. At an optical density (600nm) of 0.8 the culture was induced with 1 mMol IPTG. The cells were harvested by centrifugation for 20 min at 5000 rpm. The cells were washed with 0.9 % saline and centrifuged as above. The cells were suspended in 10 ml 50 mM phosphate buffer pH 7.5 containing 0.6 mM phenylmethylsulfonyl fluoride, cooled on ice and sonified 6 x 10 sec (Branson sonifier level 4). The suspension was centrifuged at 15000 rpm at 4 °C for 20 min. The supernatant contained about 80 mg protein and was loaded onto a HiTrap (Pharmacia) metal chelate affinity column (volume 5 ml), charged with Ni²⁺ ions. The column was washed with 50 ml of 50 mM phosphate buffer pH 7.5. The 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone-5-phosphate reductase was eluted with 10 ml of 50 mM phosphate buffer pH 7.5 containing 500 mM imidazole. The elute was analyzed on a SDS-PAGE. The desired 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone-5- phosphate reductase has a molecular weight of 27 kDa.

Screening Example 1

Screening of GTP cyclohydrolase II activity

Assay mixtures contained 100 mM Tris hydrochloride pH 8.5, 5 mM MgCI₂, 5 mM dithiothreitol, 1 mM GTP and 20 μl enzyme (80 μg) sample as given in table 1 in a total volume of 80 μl. Separately, to these mixtures 0.5 and 5.0 mM sodium pyrophosphate, 0.5 and 5.0 mM methylene-bisphosphonate was added. They were incubated at 37 °C for 30 min. A derivatization solution (20 μl) of 5 % diacetyl and 250 mM EDTA was added and each mixture incubated at 90 °C for 1 h. The diacetyl reacts with the enzyme product to yield 6,7-dimethylpterin, which was subsequently measured by reverse-phase HPLC on a column of Nucleosil RP1 8. The eluent contained 100 mM ammonium formate and 25 % methanol. The effluent was monitored fluorometrically (excitation 365 nm; emission 435 nm) . One unit of enzyme activity catalyzes the formation of 1 nmol of 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate per h at 37 °C.

Screening Example 2

Assay mixtures contained 1 00 mM Tris hydrochloride pH 8.5, 1 0 mM MgCI₂, 5 mM dithiothreitol, 0.5 mM GTP and 20 μl enzyme sample. Separately to these mixtures 0.25 and 2.5 mM 2,6-diamino-5-formamido-pyrimidinon or 0.25 and 2.5 mM 1 -hydroxy-ethylidene-1 , 1 -bis-phosphoric acid were added, and the mixtures were incubated at 37 °C for 30 min. The further steps were identical to Screening Example 1 .

Table 1

Screening Example 3

The screening Example 1 is repeated with the exception that the derivatization solution is not added. Instead the assay mixtures contains additionally 100 g 2,5-diamino-6-ribosylamino-4(3H)-pyrimidine 5'-phosphate reductase and 0.1 mM NADH and the total volume is 500 μl. The assay mixture is incubated in a cuvette at 37 °C and the absorbence at 340 nm is monitored. This gives the rate of consumed NADH which is equivalent to the rate of formed 2, 5-diamino- 6-ribosylamino-4(3H)-pyrimidine 5'-phosphate.

Screening Example 4

Assay mixture contains 1 00 mM Tris hydrochloride, pH 8.5, 5 mM MgCI₂, 5 mM dithiothreitol, 1 mM GTP and 20 μl enzyme (80 μg MBP-ribA) sample in a total volume of 80 μl. Analogous mixtures contain additionally 0.5 and 5.0 mM sodium pyrophosphate. After incubation for 30 min. at 37 °C the reaction is stopped by adding 20 μl of 50 mM EDTA. The enzyme product, 2,5-diamino-6- ribosylamino-4(3H)-pyrimidinedione 5'-phosphate is determined by HPLC with a reverse phase Nucleosil RP1 8 column. The eluent contains 0.6 % isopropanol and 1 % triethylammonium phosphate, pH 7.0. The product is identified by UV absorbance at 293 nm and quantified by integration. The extinction coefficient is 1 2, 1 00 M ¹.

Screening Example 5

Assay mixture contains 1 00 mM Tris hydrochloride, pH 8.5, 5 mM MgCI₂, 0.1 mM GTP and 20 μl enzyme (80 μg MBP-ribA) sample in a total volume of 500 μl. Analogous mixtures contain additionally 0.5 and 5.0 mM sodium pyrophosphate. The assay is performed in a quartz cuvette at 37 °C under exclusion of oxygen. The reaction rate is determined directly by monitoring the absorbance of GTP at 252 nm and of 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinedione 5'-phosphate at 293 nm. Screening Example 6

A part C containing 0 resp. 2.0 resp. 20 mM sodium pyrophosphate in 20 μl H₂O is added to a mixture A containing 400 mM Tris hydrochloride pH 8.5, 30 mM MgCI₂, 20 mM DTT and 50 μg MBPj bA enzyme in a total of 20 μl. A third solution B containing 4 mM GTP in 20 μl H₂O is added to start the reaction. The mixture is incubated at 35°C for 30 min. 20 μl of mixture E containing 5% diacetyl and 250 mM EDTA is added and heated for 1 h at 90°C. The amount of 6,7-dimethylpterin was determined fluorometrically (excitation 365 nm; emission 435 nm) by comparison with a 3 μM 6,7-dimethylpterin standard.

Screening Example 7

Screening of 3,4-dihydroxy-2-butanone 4-phoshate synthase activity. Assay mixture contains 300 mM potassium phosphate pH 7,5, 20 mM MgCI₂, 1 0 mM ribose 5-phosphate and 0.1 U pentose-phosphate isomerase in a total volume of 30 μl. They were incubated at 37 °C for 1 5 min. and 1 0 μl of the enzyme sample and 10 μl of H₂O resp. 10 l of 100 mM pyruvaldehydoxim as given in table 2 were added. The mixture (50 μl) was incubated for 1 h. A solution (50 μl) containing 200 mM potassium phosphate pH 7.5, 40 mM dithiothreitol, 50 mM EDTA, 4 mM 5-amino-6-ribitylamino-2,4( 1 H,3H)- pyrimidinedione and 10 U of the lumazine synthase/riboflavin synthase complex from Bacillus subtilis was added. The lumazine synthase/riboflavin synthase is prepared as described by Schott et al. , 1 990. The mixture was incubated 1 h at 37 °C and then denaturated at 95 °C for 5 min. Riboflavin was determined by reverse-phase HPLC on a column of Nucleosil RP1 8. The eluent contained 1 00 mM ammonium formate and 40 % methanol. The effluent was monitored fluorometrically (excitation 445 nm; emission 51 6 nm). One unit of enzyme activity catalyzes the formation of 1 nmol of 3,4-dihydroxy-2-butanone 4- phosphate per h at 37 °C. Table 2

Screening Example 8

The assay is performed as described in screening Example 7. Instead of the lumazine synthase/riboflavin synthase complex only 6,7-dimethyl-8-ribityl- lumazine synthase is added. 6,7-Dimethyl-8-ribityl-lumazine is determined by reverse phase HPLC on a column of nucleosil RP1 8. The eluent contains 10% methanol and 30 mM formic acid. The effluent is monitored fluorometrically (excitation 408 nm; emission 487 nm).

Screening Example 9

Assay mixture contains 300 mM potassium phosphate pH 7.5, 20 mM MgCI₂, 1 0 mM ribose 5-phosphate and 0.1 U pentose-phosphate isomerase in a total volume of 30 μl. They are incubated at 37 °C for 1 5 min. 10 μl of the enzyme sample (MBP-ribAS) and 10 μl of H₂O resp. 1 0 μl of 1 00 mM pyruvaldehydoxim are added. The mixture is incubated for 1 h. A solution (50 μl) containing 2 % o- phenylenediamine, 20 mM DTT and 100 mM EDTA is added and the mixture incubated at 90°C for 1 h. The o-phenylenediamine reacts with 3,4-dihydroxy- 2-butanone 4-phosphate to yield dimethylquinoxaline which is subsequently measured by reverse phase HPLC on a column of Nucleosil RP1 8. The eluent contains 1 00 mM ammonium formate and 25 % methanol. The effluent is monitored fluorometrically. Screening Example 10

A part M containing 0 resp. 60 mM pyruvaldehydoxim in 20 μl H₂O is added to a mixture H containing 400 mM potassium phosphate pH 7.5, 50 mM MgCI₂ and 50 μg MBP_ribA enzyme in a total of 20 μl. A third solution J with 5mM ribulose-5-phosphate in 20 μl H₂O is added to start the reaction. The mixture is incubated for 30 min. at 37 °C. A mixture P containing 50 mM DTT, 1 00 mM EDTA and 10 mM 5-amino-6-ribitylamino-2,4( 1 H,3H)-pyrimidinedione in a total of 20 μl and a mixture R (20 μl) containing 200 mM potassium phosphate pH 7.5 and 1 0 U of 6,7- dimethyl-8-ribityl-lumazine synthase are added. The mixture is incubated for 1 h at 37 °C and then denaturated by adding 1 00 μl solution T containing 1 5% trichloroacetic acid. The amount of 6,7-dimethyl-8- ribityl-lumazine is determined fluorometrically (excitation 408 nm; emission 487 nm) by comparison with a 1 6 μM 6,7-dimethyl-8-ribityl-lumazine standard.

Annex A References

Bacher, A. ( 1 991 ) Biosynthesis of flavins. In: Chemistry and Biochemistry of Flavoproteins (F. Mϋller, ed.) 1 , CRC Press, Boca Raton, 21 5-259.

Bacher, A., Eberhardt, S., Richter, G. ( 1 996) Biosynthesis of Riboflavin. In: Escherichia coli and Salmonella (F. Neidhardt, ed.), ASM, Washington DC, 657- 664.

Bult C.J., White O., Olsen G.J., Zhou L., Fleischmann R.D., Sutton G.G., Blake J.A., FitzGerald L.M., Clayton R.A., Gocayne J.D. , Kerlavage A.R., Dougherty B.A., Tomb J.F. , Adams M.D., Reich C. I., Overbeek R., Kirkness E.F., Weinstock K.G., Merrick J.M., Glodek A., Scott J.L., Geoghagen N.S.M., Venter J.C. ( 1 996) Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii, Science 273(5278): 1058-73.

Cole, S.T., Brosch, R., Parkhill, J., Gamier, T., Churcher, C, Harris, D., Gordon, S.V., Eiglmeier, K., Gas, S., Barry III, C.E., Tekaia, F., Badcock, K., Basham, D., Brown, D., Chillingworth, T., Connor, R., Davies, R., Devlin, K., Feltwell, T., Gentles, S., Hamlin, N., Holroyd, S., Hornsby, T., Jagels, K., Krogh, A., McLean, J., Moule, S., Murphy, L., Oliver, S., Osborne, J., Quail, M.A., Rajandream, M.A. , Rogers, J., Rutter, S., Seeger, K. , Skelton, S., Squares, S., Sqares, R., Sulston, J.E., Taylor, K., Whitehead, S., Barrell, B.G. ( 1 998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-544

Deckert, G., Warren, P.V., Gaasterland, T., Young, W.G., Lenox, A.L., Graham, D.E., Overbeek, R., Snead, M.A., Keller, M., Aujay, M., Huber, R., Feldman, R.A., Short, J.M., Olson, G.J., Swanson, R.V. ( 1 998) The complete genome of the hyperthermophilic bacterium Aquifex aeolicus. Nature 392, 353- 358.

Fleischmann, R.D., Adams M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.-F., Dougherty, B.A., Merrick, J.M., Mckenney, K., Sutton, G., Fitzhugh, W., Fields, C.A., Gocayne, J.D., Scott, J.D., Shirley, R., Liu, L.-l., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L.D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., Mcdonald, L.A., Small, K.V., Fraser, CM., Smith, H.O., Venter, J.C ( 1 995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496-51 2.

Fuller, T.E. , Mulks, M.H. (1 995) Characterization of Actinobacillus pleuropneumoniae riboflavin biosynthesis genes. J. Bact. 1 77, 7265-7270.

Gusarov, 1.1., Kreneva, R.A., Podchernyaev, D.A., lomantas, Y.V.,A balakina, E.G., Stoinova, N.V., Perumov, D.A. and Kozlov, Y. I. ( 1 997) Riboflavin biosynthesis genes of Bacillus amyloliquefaciens: primary structure, arrangement and regulation. Mol. Biol. 31 , 446-453.

Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C, Wada, T., Watanabe, A., Yamada, M., Yasuda, M. and Tabata, S. ( 1 996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3 (3), 109-1 36.

Katzenmeier G. ( 1 991 ) Klonierung und molekularbiologische Charakterisierung des Gens fur GTP-Cyclohydrolase I aus Escherichia coli. Doctor thesis, TU Mϋnchen, Germany.

Klenk, H.P., Clayton, R.A., Tomb, J., White, O., Nelson, K.E., Ketchum, K.A., Dodson, R.J., Gwinn, M., Hickey, E. K., Peterson, J.D., Richardson, D.L., Kerlavage, A. R., Graham, D.E., Kyrpides, N.C, Fleischmann, R.D., Quackenbush, J., Lee, N.H., Sutton, G.G., Gill, S., Kirkness, E.F., Dougherty, B.A., McKenney, K., Adams, M.D., Loftus, B., Peterson, S., Reich, C.I., McNeil, L.K., Badger, J.H., Glodek, A., Zhou, L., Overbeek, R., Gocayne, J.D., Weidman, J.F., McDonald, L., Utterback, T., Cotton, M.D., Spriggs, T., Artiach, P., Kaine, B.P., Sykes, S.M., Sadow, P.W., D'Andrea, K.P., Bowman, C, Fujii, C, Garland, S.A. , Mason, T.M., Olsen, G.J., Fraser, CM., Smith, H.O., Woese, C.R., Venter, J.C. ( 1 997) The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus. Nature 390, 364-370.

Kobayashi, M., Sugiyama, M., Yamamoto, K. ( 1 995) Isolation of cDNA's encoding GTP cyclohydrolase II from Arabidobsis thaliana. Gene 1 60, 303-304

Lee, C.Y., O'Kane, D.J., Meighen, E.A. ( 1 994) Riboflavin synthesis genes are linked with the lux operon of Photobacterium phosphoreum. J. Bacteriol. 1 76, 2100-2104.

Liauta-Teglivets, O.Y., Hasslacher, M., Boretsky, Y.R. , Kohlwein, S.D., Shavlovskii, G.M. ( 1 995) Molecular cloning of the GTP-cyclohydrolase structural gene RIB1 of Pichia guilliermondii involved in ribovlavin biosynthesis. Yeast 1 1 , 945-952.

Newman, T., de Bruijn, F. J., Green, P., Keegstra, K., Kende, H., Mclntosh, L., Ohlrogge, J., Raikhel, N., Somerville, S., Thomashow, M., Retzel, E., Somerville, C. ( 1 994) Genes galore: a summary of methods for accessing results from large-scale partial sequencing of anonymous Arabidopsis cDNA clones. Plant Physiol. 106, 1 241 -1 255.

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Annex B

Description GTP cyclohydrolase II/3, 4-dihydroxy-2-buranone- 4-phosphate synthase

Organism Arabidopsis thaliana

10- 20 30 40 50 60

I I I I I I

ATGTCTTCCATCAATTTATCCTCATCATCTCCTTCCACCATCTCTCTTTCCCGCTCAAGA

M S S I N L S S S S P S T I S L S R S R

70 80 90 100 110 120

I I I I I I

CTAAGCCAAAGCTCTACTACATTACTCCATGGACTACATCGTGTTACTTTACCATCTAAC S Q S S T T L L H G L K R V T L P S N

130 140 150 160 170 150

I I I I I I

CATCCATTGTCSA.CCTTTTCCATTAAAACCAATACTGGAAAAGTTAAGGCTGCAGTGATC

H P L S T F S I K T N T G K V K A A V I

190 200 210 220 230 240

I ! 1 I I 1

TCTAGAGAAGATGATCTGCTCTCATTCACCAACGGAAACACTCCTCTCTCAAATGGGTCT

S R E D D L L S F T N G N T P L S N G S

250 260 270 280 290 300

1 ! I I I I

CTCAΓTGATGATCGGACCGAAGAGCCATTAGAGGCTGATTCGGTTTCACTTGGAACACTT L I D D R T E E P L E A D S V S L G T L

310 320 330 340 350 360

I I 1 I I I

GCTGCTGATTCTGCTCCTGCACCAGCCAATGGTTTTGTTGCTGAAGATGATGACTTTGAG

A A D S A P A P A N G F E D D D F E

370 380 390 400 410 420

I I I 1 1 I

TTGGATTTACCAACTCCTGGTTTCTCTTCTATCCCCGAGGCCATTGAAGATATACGCCAA

L D L P T P G F S S I P E A I E D I R Q Annex B (cont.)

430 440 450 460 470 480

I I I I I I

GGAAAGCTTGTGGTGGTTGTGGATGATGAAGATAGGGAAAATGAAGGGGATTTGGTGATG

G K V V V V D D E D R E N E G D L V M

490 500 510 520 530 540

I I I I I I

GCTGCTCAGTTAGCAACACCTGAAGCTATGGCTTTTATTGTGAGACATGGAACTGGGATA

A A Q L A T P E A M A F I V R H G T G I

550 560 570 580 590 600 I I I 1 1 I

GTTTGTGTGAGCATGAAAGAAGATGATCTCGAGAGGTTGCACCTTCCTCTAATGGTGAAT

V C V S M E D D L E R L H L P L M V N

610 620 630 640 650 660

I I 1 I I I

CAGAAGGAAAACGAAGAAAAGCTCTCTACTGCATTTACAGTGACTGTGGATGCAAAACAT

Q K E N E E K L S T A F T V T V D A K H

670 680 690 700 710 720 I I I I I I

GGTACAACAACGGGAGTATCAGCTCGTGACAGGGCAACAACCATATTGTCTCTTGCATCA G T T T G V S A R D R A T T I L S L A S

730 740 750 760 770 780

I I I I i I

AGAGATTCAAAGCCTGAGGATTTCAATCGTCCAGGTCATATCTTCCCACTGAAGTATCGG

R D S K P E D F N R P G H I F P L K Y R

790 800 810 820 830 840

I I 1 I 1 I

GAAGGTGGGGTTCTGAAAAGGGCTGGACACACTGAAGCATCTGTTGATCTCACTGTTTTA

E G G V L K R A G H T E A S V D T V

850 860 870 880 890 900

I I I I I I

GCTGGACTGGATCCTGTTGGAGTACTTTGTGAAATTGTTGATGATGATGGTTCCATGGCT

A G L D P V G V C E I V D D D G S A Annex B (cont.)

910 920 930 940 950 960

I I I I I I

AGATTACCAAAACTTCGTGAATTTGCCGCCGAGAACAACCTGAAAGTTGTTTCCATCGCA

R L P K L R E F A A E N N L K V V S I A

970 980 990 1000 1010 1020

1 I I I I I

GATTTGATCAGGTATAGAAGAAAGAGAGATAAATTAGTGGAACGTGCTTCTGCGGCTCGG

D L I R Y R R K R D K L V E R A S A A R

1030 1040 1050 1060 1070 1080

I I I I I I

ATCCCAACAATGTGGGGACCTTTCACTGCTTACTGCTATAGGTCCATATTAGACGGAATA

I P T M W G P F T A Y C Y R S T L D G I

1090 1100 1110 1120 1130 1140 I I I I I 1

GAGCACATAGCAATGGTTAAGGGTGAGATTGGTGACGGTCAAGACATTCTCGTGAGAGTT E H I A M V K G E I G D G Q D I L V R V

1150 1160 1170 1180 1190 1200

I I I I 1 I

CATTCTGAATGTCTAACAGGGGACATATTTGGGTCTGCAAGGTGTGATTGCGGGAACCAG

H S E C L T G D I F G S A R C D C G N Q

1210 1220 1230 1240 1250 1260

I I I I I I

CTAGCACTCTCGATGCAGCAGATCGAGGCTACTGGTCGCGGTGTGCTGGTTTACCTACGT

L A L S M Q Q I E A T G R G V L V Y L R

1270 1280 1290 1300 1310 1320 I 1 I I I I

GGACATGAAGGAAGAGGGATCGGTTTAGGACACAAGCTTCGAGCTTACAATCTGCAAGAT G H E G R G I G L G H K R A Y N L Q D

1330 1340 1350 1360 1370 1380

I I I I I I

GCTGGTCGAGACACGGTTGAAGCTAATGAGGAATTAGGACTTCCTGTTGATTCTAGAGAG

A G R D T V E A N E E L G P V D S R E Annex B (cont.)

1390 1400 1410 1420 1430 1440

I I I I I I

TATGGAATTGGTGCACAGATAATAAGGGATTTAGGTGTTAGGACAATGAAGCTGATGACA

Y G I G A Q I I R D L G V R T M K M T

1450 1460 1470 1480 1490 1500

I I I I I I

AATAATCCCGCAAAGTATGTTGGTTTGAAGGGATATGGATTAGCCATTGTTGGGAGAGTC

N N P A Y V G L K G Y G L A I V G R V

1510 1520 1530 1540 1550 1560 I I I I I I

CCTCTATTGAGTCTTATCACGAAGGAGAATAAGAGATATCTGGAGACAAAGCGGACCAAG P L L S L I T K E N R Y L E T K R T K

1570 1580 1590 1600 1610 1620

I I I I I I

ATGGGTCACATGTATGGCTTGAAGTTCAAAGGGGATGTTGTGGAGAAGATTGAGTCTGAA

M G H M Y G L K F K G D V V E K I E S E

1630 I TCTGAGTCCTAA

S E S -

Annex C

Description GTP cyclohydrolase II/3, 4-dihydroxy-2-butanone- -phosphate synthase

Organism. Lycoper3icon esculentum

10 20. 30 40 50 60

I I I 1 I I

ATGGCTTCCATCAACATTTCTTTCCCTTCAACAACATTTCACAGTCGATCACAAGCGAAC

M A S I N I S F P S T T F H S R S Q A N

70 80 90 100 110 120

I I I I I I

TTCAAGTTGTTCAGTGGGTTGCATTCTGGAGATGTAGTTTCTCTTAATGGGGTCTCGTCA

F K L F S G L H S G D V V S N G V S S

130 140 150 160 170 180

I I I I 1 I

GGGTCATTCATACGGCTTAATGCTAGACCACACTTAACCATCAAGAGTGATTTCAAGATT

G S F I R L N A R P H L T I S D F K I

190 200 210 220 230 240

I I I I I I

ACTTCTGCATTGTTATCTGGTGAAGGAGACATCCGATTTCAATCCAGGGGAGAAAAAACT

T S A L L S G E G D I R F Q S R G E K T

250 260 270 280 290 300

I I I I I I

CTGTTTAGTAGCCTCTCTACAGGAACTGAGACTCAACCTGATGCTGTAACCTTTGCAACG

L F S S L S T G T E T Q P D A V T F A T

310 320 330 340 350 360

I I I I I I

CTAGAAGCAGATGGTGTTCCTACAACTAGTGGTTTTCTTTCAGATGACGATGAATGTGAT

L E A D G V P _. T T S G F L S D D D E C D

370 380 390 400 410 420

I I I I I I

TTGGATCGACCGACCGAAGGTTTTTCATCTGTTCCTGAGGCTATTGAGGACATTCGCCAA

L D R P T E G F S S V P E A I E D I R Q

430 440 450 460 470 480 Annex C (cont.)

I I I I I I

GGAAAGATGGTGCTAGTTACAGATGATGAAGACAGAGAAAATGAAGGGGATTTAGTTATG G K M V L V T D D E D R E N E G D L V M

490 500 510 520 530 540 I I I I I I

GCTGCATCCAAAGCTACACCAGAAGCAATGGCTTTCTTTGTGAAGTATGGAACAGGGATA

A A S K A T P E A M A F F V K Y G T G I

550 560 570 580 590 600 I I I I I I

GTTTGTGTGAGCATGACAGAAGAACACTTGGAGAGGCTACAGCTTCCGTTGATGGTAAAC

V C V S M T E E H L E R L Q L P L M V N

610 620 630 640 650 660 I I I I I I

GATAAAAAGAATGAGGAGAAACTTTGTACAGCATTCACAGTCTCAGTGGATGCGAAACAT

D K K N E E K L C T A F T V S V D A K H

670 680 690 700 710 720 I I I I I I

GGAACGACTACAGGAGTATCTGCTCATGATAGAGCAACGACAGTATTGGCACTTGCGTCC

G T T T G V S A H D R A T T V L A L A S

730 740 750 760 770 780

I I I I I I

GGAGATTCAAAGCCTGAGGATTTCAATCGACCGGGACATATCTTTCCTTTGAAATACAGG

G D S K P E D F N R P G H I F P L K Y R

790 800 810 820 830 840 I I I I I I

GAAGGCGGGGTTTTAAAACGAGCTGGACATACTGAGGCTTCTGTGGATCTTGCCGTGTTA E G G V K R A G H T E A S V D A V L

850 860 870 880 890 900

I I I I I I

GCTGGCTTAGACCCTGTTGGAGTAATATGTGAGGTTGTGGATGATGATGGTTCCATGGCT

A G L D P V G V I C E V V D D D G S M A

910 920 930 940 950 960 Annex C (cont.)

I I I I I I

AGATTGCCTATGCTTCGTCAATTTGCAAAAGAACATAACTTGAAGATCATATCAGTTGCT R L P M L R Q F A K E H N L K I I S V A

970 980 990 1000 1010 1020

I I I I I I

GACTTAATCAGATATAGGAGAAAAACAGATCAGCTGGTAGAGCATGCTTCTGCTGCAAGA

D L I R Y R R K T D Q L V E H A S A A R

1030 1040 1050 1060 1070 1080

I I 1 I I I

ATACCTACAATGTGGGGCCCGTTTACTGCCCACTGCTTCAAGTCAATAATAGATGGAATT

I P T M G P F T A H C F K S I I D G I

1090 1100 1110 1120 1130 1140 I I I I I I

GAGCACATTGCTATGGTTAAGGGGGATATAGGAGATGGACAAGATATTCTTGTTCGGGTA E H I A M V G D I G D G Q D I L V R V

1150 1160 1170 1180 1190 1200

I 1 I I I I

CACTCGGAATGCCTCACAGGAGATATATTTGGTTCAGCCAGATGTGACTGTGGATCCCAG

H S E C L T G D I F G S A R C D C G S Q

1210 1220 1230 1240 1250 1260

I I I I I I

CTGGCAACTGCAATGAAGCAAATTGAGGCTGCTGGCAGGGGGGTTTTGGTTTACCTCCGC

L A T A M K Q I E A A G R G V L V Y R

1270 1280 1290 1300 1310 1320

I I I I I I

GGTCATGATGGTAGAGGCATTGGTTTGGGTCACAAACTTCGTGCTTATAATTTACAAGAT

G H D G R G I G L G H K L R A Y N L Q D

1330 1340 1350 1360 1370 1380

I I I I I I

GCTGGACGTGATACTGTTGAAGCGAATGAAGATCTTGGTTTGCCCGTTGATTCAAGAGAG

A G R D T V E A N E D L G L P V D S R E

1390 1400 1410 1420 1430 1440 Annex C (cont.)

I I I I I I

TATGGGGTTGGTGCACAGATCTTAAGGGATCTTGGTGTTCGAACTATGAAGTTTATAACG Y G V G A Q I L R D L G V R T M K F 1 T

1450 1460 1470 1480 1490 1500

I I I I I I

AACAATCCTGCACAATACAGCTGGCTAAAAGGTTATGGTTTGGCAATTTCTGGTATGGTC

N N P A Q Y S W L K G Y G L A I S G M V

1510 1520 1530 1540 1550 1560 I I I I I I

CCCGTTGTTACTCCCTTTACTAACCACTACGACACATATTTGGAAACAACACGAGCTAAA

P V V T P F T N H Y D T Y L E T T R A K

1570 1580 1590 1600 1610 1620

I I I I I I

ATGGGCCATGTATACGGCTTGAACCTTATTCGCCCGGCAACCAGCACCAGCACAACAAAT

M G H V Y G L N L I R P A T S T S T T N

1630 1640 1650

I 1 I

GGTAAACCAAACTCTGAGAATACTTCTACTATAAGATGA

G K P N S E N T S T I R -

Annex D

Description 2, 5-diamino-6-ribosylamino-4 (3H) -pyrimidinone reductase Organism Saccharomyces cerevisiae

10 20 30 40 50 60

I I. I I I I

ATGTCTTTGACACCACTGTGTGAAGATTTACCACAATTTCTGCAAAACTATCTACCGAAT

M S L T P C E D P Q F L Q N Y L P N

70 80 90 100 110 120

I I I I I I

GCTGGTCAAACGGAAAATACCATTGTGCCCTTTGTCACACTAACTTATGCTCAATCGCTC

A G Q T E N T I V P F V T L T Y A Q S L

130 140 150 160 170 180 I I I I I I

GACGCGAGAGTATCTAGGGGCCCTGGAGTGAGGACTACAATTTCACATCCCGAGACCAAA D A R V S R G P G V R T T I S H P E T K

190 200 210 220 230 240

I I I I I I

ACAATGACGCATTATTTGAGACATCATCACGATGGAATACTCGTAGGAAGTGGAACAGTG

T M T H Y L R H H H D G I L V G S G T V

250 260 270 280 290 300

I I I I I I

CTAGCTGATAATCCTGGATTGAATTGTAAATGGGGTCCCGATCCGGCTGCAAATTCCCCA

L A D N P G L N C K W G P D P A A N S P

310 320 330 340 350 360

I I I I I I

AGGCCAATAATAATAGATACAAAGCAAAAGTGGCGATTTGATGGTTCAAAAATGCAAGAA

R P I I I D T K Q K R F D G S K M Q E

370 380 390 400 410 420

I I I I I I

CTTTTTATTAAACGACAGGGTAAGCCGCCAATCGTTGTTGTCACAAGTGAGCCCATTATA

L F I K R Q G K P P I V V V T S E P I I

430 440 450 460 470 480 I I Annex D (cont.)

AAAGAACAACATGTAGACTACGCAATTTGTCCAΆTAAATGATACTACGAAATTGGTCGAT K E Q H V D Y A I C P I N D T T K L V D

490 500 510 520 530 540

I I I I I I TGGAAGAAATTGTTTGAGATATTAAAAGAAGAATTCAATATAAGGTCAGTAATGGTTGAA K K L F E I L K E E F N I R S V M V E

550 560 570 580 590 600

I I I I I I

GGAGGTGCCAATGTAATAAATCAGTTGTTGCTGAGGAGCGATATTGTCAACAGTCTTATA

G G A N V I N Q L L L R S D I V N S I

610 620 630 640 650 660

I I I I I I

ATAACTATTGGATCAACATTTCTGGGTAGCTCAGGCACCGAAGTTAGCCCACCCCAAACA

I T I G S T F L G S S G T E V S P P Q T

670 680 690 700 710 720 I I I I I I

GTAAATTTAAAGGATATGTCATGGTGGAAGGGCATTACCGATGTGGTGCTTTGTGCGAGA V N L K D M S K G I T D V V L C A R

730

I CTGGCCGATGACTAA

L A D D -

Claims

1. A method for screening for the presence or absence of inhibition of GTP cyclohydrolase II activity, comprising the following steps:

(b) reacting said first mixture during a predetermined period of time at a predetermined temperature and. subsequently detecting the level of 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5'-phosphate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a chemical test sample

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting the level of 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5'-phosphate,

(e) determining the presence of inhibition of GTP cyclohydrolase II by observation of whether the level detected in step (d) is lower than the level detected in step (b).

2. A method for screening for the presence or absence of inhibition of GTP cyclohydrolase II activity, comprising the following steps:

(b) reacting said first mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5'-phosphate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a specific inhibitor for said GTP cyclohydrolase II,

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting the level of 2,5-diamino-6-ribosylamino-4(3H)- pyrimidinone 5'-phosphate, (e) determining the presence of resistance to inhibition of GTP cyclohydrolase II by observation of whether the level detected in step (d) is similar to the level detected in step (b).

3. The method according to claims 1 or 2, wherein said aqueous mixture contains a bivalent metal ion.

4. The method according to claim 3, wherein said bivalent metal ion is the magnesium ion.

5. The method according to claims 1 or 2, wherein said mixture contains an antioxidative substance.

6. The method according to claims 1 or 2, wherein said mixture has a pH in the range of 6 to 9.5.

7. The method according to claims 1 or 2, wherein a premixture is prepared which lacks one essential ingredient and the reaction is started by adding said ingredient.

8. The method according to claims 1 or 2 characterized in that the level of 2,5-diamino-6-ribosyIamino-4(3H)-pyrimidinon 5'-phosphate is detected directly or after chemical or enzymatic derivatization.

9. The method according to claims 1 and 8 or 2 and 8, wherein said direct detection is carried out after the reaction has been terminated by adding a chelating agent for said bivalent metal ion.

10. The method according to claims 1 or 2, wherein the chemical derivatization is carried out by adding a vicinal dioxo compound.

11. The method according to claim 10, wherein said dioxo compound is diacetyl.

12. The method according to claims 1 or 2, wherein the enzymatic detection is carried out by adding 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase and NAD(P)H and detecting the formation of NAD(P).

13. A method for screening for the presence or absence of inhibition of 3,4- dihydroxy-2-butanone 4-phosphate synthase activity, comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence and ribulose 5-phosphate,

(b) reacting said mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 3,4-dihydroxy-2-butanone 4-phosphate,

(c) preparing a second aqueous mixture by including in said first mixture a predetermined amount of a specific inhibitor for said 3,4-dihydroxy-2-butanone 4-phosphate synthase,

(d) reacting said second mixture during the predetermined period of time at the predetermined temperature and subsequently detecting again the level of 3,4-dihydroxy-2-butanone 4- phosphate,

(e) determining the presence of inhibition of 3,4-dihydroxy-2- butanone 4-phosphate synthase by observation of whether the level detected in step (d) is lower than the level detected in step (b).

14. A method for screening for the presence or absence of inhibition of 3,4- dihydroxy-2-butanone 4-phosphate synthase activity, comprising the following steps:

(a) preparing a first aqueous mixture containing a protein having a mutated plant-type 3,4-dihydroxy-2- butanone 4-phosphate synthase sequence and ribulose-5- phosphate, (b) reacting said mixture during a predetermined period of time at a predetermined temperature and subsequently detecting the level of 3,4-dihydroxy-2-butanone 4-phosphate,

(e) determining the presence of resistance to inhibition of 3,4- dihydroxy-2-butanoπe 4-phosphate synthase by observation of whether the level detected in step (d) is similar to the level detected in step (b).

15. The method according to claims 13 or 14, wherein said aqueous mixture contains a bivalent metal ion.

16. The method according to claim 15, wherein said bivalent metal ion is the magnesium ion.

17. The method according to claims 13 or 14, wherein said mixture has a pH in the range of 6 to 9.5.

18. The method according to claims 13 or 14, wherein a premixture is prepared which lacks one essential ingredient and the reaction is started by adding said ingredient.

19. The method according to claims 13 or 14, wherein the presence of 3,4- dihydroxy-2-butanone 4-phosphate is detected directly or after chemical or enzymatic derivatization.

20. The method according to claim 19, wherein said detection is carried out after the reaction has been terminated by adding a chelating agent for said bivalent metal ion.

21. The method according to claim 19, wherein said chemical derivatization is carried out by adding an aromatic or heteroaromatic ortho-diamine.

22. The method according to claim 20, wherein for enzymatic derivatization 6,7-dimethyl-8-ribityllumazine synthase and 5-amino-6-ribitylamino- 2,4(1 H,3H)-pyrimidinedione are added and the level of 6,7-dimethyl-8- ribityl-lumazine is detected.

23. The method according to claim 20, wherein for enzymatic derivatization 6,7-dimethyl-8-ribityllumazine synthase, riboflavin synthase and 5-amino- 6-ribitylamino-2,4(1 H,3H)-pyrimidinedione are added and the level of riboflavin is detected.

24. The method according to claims 13 or 14 characterized in that said first aqueous mixture is prepared by pre-reacting a first pre-mixture containing pentose-phosphate isomerase and ribose 5-phosphate and mixing said pre-reacted first pre-mixture with a second pre-mixture containing said protein having the 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence or said protein having said mutated 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence.

25. Kit of parts for screening for the presence or absence of inhibition of GTP cyclohydrolase II activity in a chemical test sample, comprising a part A with a protein having a GTP cyclohydrolase II sequence, and a part B with GTP.

26. Kit of parts for screening for the presence or absence of resistance of a protein having a mutated GTP cyclohydrolase II sequence to a specific inhibitor for GTP cyclohydrolase II activity comprising a part C with said specific inhibitor, and a part B with GTP.

27. Kit of parts according to one of claims 25 or 26, additionally comprising a bivalent metal ion in at least one of parts A, B or C or in a separate part D.

28. Kit of parts according to claim 27 wherein a chelating agent for said bivalent metal ion is contained in a separate part E.

29. Kit of parts according to one of claims 25 to 28 additionally comprising a vicinal dioxo compound either in part E or in a separate^'part F.

30. Kit of parts according to one of claims 25 to 28 additionally comprising 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase in at least one of parts A, B, C, D, E and/or a separate part G; and NAD(P)H in at least one of part A, B, C, D, E, G.

31. Kit of parts for screening for the presence or absence of inhibition of 3,4- dihydroxy-2-butanone 4-phosphate synthase activity in a chemical test sample, comprising a part H with a protein having a 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence and either a part J with ribulose 5-phosphate or a part K with pentose-phosphate isomerase in conjunction with a part L with ribose 5-phosphate.

32. Kit of parts for screening for the presence or absence of resistance of a protein having a mutated 3,4-dihydroxy-2-butanone 4-phosphate synthase sequence to a specific inhibitor for 3,4-dihydroxy-2-butanone 4- phosphate synthase activity comprising a part M with said specific inhibitor and either a part J with ribulose 5-phosphate or a part K with pentose-phosphate isomerase in conjunction with a part L with ribose 5-phosphate.

33. Kit of parts according to one of claims 31 to 32, additionally comprising a bivalent metal ion in at least one of parts H, J, K, L, M or in a separate part N.

34. Kit of parts according to claim 33, wherein a chelating agent for said bivalent metal ion is contained in a separate part P.

35. Kit of parts according to one of claims 31 to 34 additionally comprising an aromatic or heteroaromatic ortho-diamine either in part P or in a separate part Q.

36. Kit of parts according to one of claims 31 to 34, additionally comprising 6,7-dimethyl-8-ribityllumazine synthase in at least one of parts H, J, K, L, M, N, P or in a separate part R and 5-amino-6-ribitylamino-2,4(1H,3H)- pyrimidinedione in at least one of parts H, J, K, L, M, N, P, R.

37. Kit of parts according to claim 36 comprising additionally riboflavin synthase in at least one of parts H, J, K, L, M, N, P, R or in a separate part S.

38. Isolated protein having a plant enzyme sequence of GTP cyclohydrolase II and/or 3,4-dihydroxy-2-butanone 4-phosphate synthase and having the function of GTP cyclohydrolase and/or 3,4-dihydroxy-2-butanone-4- phosphate synthase.

39. Protein according to claim 38 having additionally an auxiliary protein sequence.

40. Protein according to claim 38 wherein the plant is a monocotyledonous or dicotyledonous plant.

41. Isolated DNA coding exclusively for a protein in accordance with claim

38 and optionally at least one other enzyme of the flavin biosynthesis pathways.

42. A vector comprising the nucleotide sequence of the DNA in accordance with claim 41.

43. A method of inhibiting an enzyme with GTP cyclohydrolase II activity and 3,4-dihydroxy-2-butanone 4-phosphate synthase activity of or in a plant by treatment with a compound selected from the group of chemical compounds that exhibit inhibition in the method of claims 1 or 13.

44. A method of inhibiting an enzyme with GTP cyclohydrolase II activity of or in a microorganism by treatment with a compound selected from the group of chemical compounds that exhibit inhibition in the method of claim 1.

45. A method of inhibiting an enzyme with 3,4-dihydroxy-2-butanone 4- phosphate synthase activity of or in a microorganism by treatment with a compound selected from the group of chemical compounds exhibiting inhibition in the method of claim 13.