Biosynthesis of Riboflavin

STUDIES ON THE MECHANISM OF GTP CYCLOHYDROLASE II*

Harald Ritz, Nicholas Schramek, Andreas BracherDagger, Stefan Herz§, Wolfgang Eisenreich, Gerald Richter, and Adelbert Bacher

From the Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstrasse 4, D-85747 Garching, Germany

Received for publication, January 26, 2001, and in revised form, April 6, 2001

    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GTP cyclohydrolase II catalyzes the first committed reaction in the biosynthesis of the vitamin riboflavin. The recombinant enzyme from Escherichia coli is shown to produce 2,5-diamino-6-beta -ribosylamino-4(3H)-pyrimidinone 5'-phosphate and GMP at an approximate molar ratio of 10:1. The main product is subject to spontaneous isomerization affording the alpha -anomer. 18O from solvent water is incorporated by the enzyme into the phosphate group of the 5-aminopyrimidine derivative as well as GMP. These data are consistent with the transient formation of a covalent phosphoguanosyl derivative of the enzyme. Subsequent ring opening of the covalently bound nucleotide followed by hydrolysis of the phosphodiester bond could then afford the pyrimidine type product. The hydrolysis of the phosphodiester bond without prior ring opening could afford GMP. The enzyme reaction is cooperative with a Hill coefficient of 1.3. Inhibition by pyrophosphate is competitive. Inhibition by orthophosphate is partially uncompetitive at low concentration and competitive at concentrations above 6 mM.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

GTP cyclohydrolases represent a family of enzymes using GTP as starting material for the biosynthesis of various heterocyclic coenzymes and nucleotide analogs. Specifically, GTP cyclohydrolase I catalyzes a ring expansion of GTP (Compound 1) conducive to the formation of dihydroneopterin triphosphate (Compound 3) (1, 2) that serves as the first committed intermediate in the biosynthetic pathways of tetrahydrofolate in plants and certain microorganisms (3) and tetrahydrobiopterin in animals (4). On the other hand, GTP cyclohydrolase II catalyzes the formation of a pyrimidine derivative that serves as the first committed intermediate in the biosynthetic pathway of riboflavin (Compound 5) (Fig. 1) (5).

The structure and reaction mechanism of GTP cyclohydrolase I have been studied in considerable detail (6-11). By comparison, little is known about GTP cyclohydrolase II. Foor and Brown (5, 12) showed that the enzyme converts GTP (Compound 1) into a mixture of pyrophosphate, formate, and a heterocyclic reaction product that was assigned as 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-monophosphate (Compound 4) (Fig. 1) on the basis of indirect evidence. More recently, Blau et al. (13) obtained chromatographic evidence for the formation of two different heterocyclic products, but these were not investigated in more detail.


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Fig. 1.   Reactions catalyzed by GTP cyclohydrolase I (A) and GTP cyclohydrolase II (B).

The ribA gene specifying GTP cyclohydrolase II of Escherichia coli has been cloned by marker rescue (14). Certain microorganisms and plants were subsequently shown to specify bifunctional enzymes comprising GTP cyclohydrolase II and 3,4-dihydroxy-2-butanone 4-phosphate synthase domains (15).

The enzymes of the riboflavin biosynthetic pathway are potential targets for anti-infective chemotherapy because enterobacteria and certain yeasts are unable to absorb riboflavin from the environment and therefore absolutely depend on endogenous biosynthesis of the vitamin. The need for the development of novel antimicrobial strategies is urgent in light of the rapidly progressing resistance development in all major pathogen groups. With this aim, we describe studies on the reaction mechanism of GTP cyclohydrolase II, the first enzyme of the riboflavin biosynthetic pathway.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- H218O (18O enrichment > 95%) was purchased from Promochem (Wesel, Germany). [ribosyl-13C5]GTP was prepared as described previously (9).

Protein Purification-- Recombinant GTP cyclohydrolase II from E. coli was purified as described previously (16).

Assay of GTP Cyclohydrolase II Activity-- Assay mixtures contained 50 mM Tris hydrochloride, pH 8.5, 5 mM MgCl2, 1 mM GTP, and protein. The samples were incubated at 37 °C, and absorbance at 293 nm was recorded. The concentration of 2,5-diamino-6-beta -ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 4) was estimated photometrically using an absorption coefficient of epsilon 293 nm = 9.6 mM-1 cm-1 (17). Alternatively, enzyme assays were performed as described previously (16).

NMR Spectroscopy-- 13C and 31P NMR spectra were recorded with a DRX 500 spectrometer equipped with four channels and a pulsed gradient unit from Bruker Instruments (Karlsruhe, Germany). One-dimensional 13C and 31P NMR spectra were measured under 1H decoupling (WALTZ16). Two-dimensional HMQC and HMQC-total correlation spectroscopy spectra were performed with XWINNMR software (Bruker Instruments, Karlsruhe, Germany). The length of total correlation spectroscopy mixing was 60 ms. 13C and 1H NMR chemical shifts were referenced to external trimethylsilyl propane sulfonate. 31P chemical shifts were referenced to external 75% phosphoric acid in 10% D2O.

18O Incorporation Experiments-- Reaction mixtures contained 50 mM Tris hydrochloride, pH 8.5, 5 mM MgCl2, 5 mM dithiothreitol, 5 mM GTP, 10% D2O, and 43% H218O (>95 atom %) (v/v). The total volume was 500 µl. A solution containing 50 µg of recombinant GTP cyclohydrolase II from E. coli was added, and the mixture was incubated at 37 °C for 5 h. The reaction was terminated by the addition of EDTA to a final concentration of 10 mM and analyzed by NMR spectroscopy.

Steady-state Kinetics-- Reaction mixtures containing 10 mM Tris hydrochloride, pH 8.5, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.95 mM GTP, and 5 µg of recombinant GTP cyclohydrolase II of E. coli were incubated under anaerobic conditions at 37 °C. Aliquots of 30 µl were retrieved at intervals. After the addition of 10 µl of 40 mM EDTA solution, 30-µl aliquots were applied to an HPLC column of Hypersil ODS (5 µm, 4 × 250 mm, Schambeck, Bad Honnef, Germany) that was developed with a mixture of isopropanol/triethylamin/85% phosphoric acid/water (8:10:3:979, v/v, pH 7.0). The flow rate was 1 ml min-1. The effluent was monitored photometrically with a diode array photometer.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The product of GTP cyclohydrolase II can easily decompose under the formation of 2,4,5-triamino-4(3H)-pyrimidinone as shown already by Foor and Brown (5). To minimize decomposition, we analyzed crude reaction mixtures by 1H, 13C, and 31P NMR spectroscopy without any pretreatment. [ribosyl-13C5]GTP was used as substrate to increase the sensitivity and selectivity of NMR detection.

As a consequence of the 13C-labeling pattern of the proffered substrate, only product carbon atoms originating from the carbohydrate side chain of the substrate afforded intense 13C NMR signals (Fig. 2, Table I). These signals are all characterized by complex multiplet structures caused by 13C13C coupling. Two-dimensional 1H13C correlation experiments (Fig. 3) identified two spin systems corresponding to two major reaction products (Compounds 4 and 6, Fig. 4). The relative NMR signal intensities indicated a molar ratio of 1.4 for Compound 4/Compound 6. The 1H and 13C NMR chemical shifts, as well as 13C13C and 13C1H coupling patterns of both products were typical for N-ribosyl moieties (Table I). The glycosidic carbon atoms with 13C chemical shifts at 84.7 and 81.5 ppm correlated to 1H signals at 5.82 and 5.58 ppm, respectively, in the HMQC experiment (Fig. 3A). 13C31P couplings were observed for the 13C NMR signals of 4'- and 5'-carbon atoms of both glycosides indicating 5'-phosphate motifs (Fig. 2, Table I).


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Fig. 2.   13C NMR signals of the product mixture obtained by treatment of [ribosyl-13C5]GTP with GTP cyclohydrolase II. The reaction mixture contained 50 mM potassium phosphate, pH 8.0, 5 mM MgCl2, 10 mM dithiothreitol, and 5 mM [ribosyl-13C5]GTP. It was incubated at 37 °C for 1 h. EDTA was added to a final concentration of 10 mM to terminate the enzyme reaction. The reaction mixture was analyzed by NMR spectroscopy without prior purification. Coupling patterns for Compounds 6 and 4 are indicated by alpha  and beta , respectively.

                              
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Table I
NMR data of [13C5-ribosyl]2,5-diamino-6alpha -ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 6) and [13C5-ribosyl]2,5-diamino-6beta -ribosylamino-4(3H)-pyrimidinone 5'-phosphate (Compound 4)


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Fig. 3.   Two-dimensional 1H13C correlation spectra of a product mixture obtained by treatment of [ribosyl-13C5]GTP with GTP cyclohydrolase. A, HMQC; B, HMQC-total correlation spectroscopy. One-dimensional 13C NMR spectra are projected in the 13C dimension. For other details see the Fig. 2 legend.


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Fig. 4.   Isomerization of the GTP cyclohydrolase II reaction product.

The 31P NMR spectra of reaction mixtures obtained with unlabeled GTP as substrate showed three singlets at 7.07, 7.13 (Compound 4), and 7.22 ppm (Compound 6), reflecting three phosphomonoesters and a broad signal at -3.75 ppm, reflecting inorganic pyrophosphate. Signal integrals are summarized in Table II. The low intensity signal at 7.07 ppm was tentatively interpreted as GMP. This assignment was subsequently confirmed by HPLC analysis (Table III, Fig. 5). The rate of formation of GMP as compared with that of the main products, Compounds 4 and 6, is about 10-15% as shown by HPLC and 31P NMR analysis.

                              
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Table II
31P NMR signals of the GTP cyclohydrolase II product mixture

                              
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Table III
HPLC analysis of reaction mixtures
Samples were applied to a HPLC column of Hypersil ODS (5 µm, 4 × 250 mm) that was developed with a mixture of isopropanol/triethylamin/85% phosphoric acid/water (8:10:3:979, v/v, pH 7.0) at a flow rate of 1 ml min-1.


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Fig. 5.   Steady-state kinetics of GTP cyclohydrolase II. The reaction mixture contained 10 mM Tris hydrochloride, pH 8.5, 5 mM MgCl2, 0.5 mM dithiothreitol, 0.95 mM GTP, and 5 µg of recombinant GTP cyclohydrolase II of E. coli. It was incubated under anaerobic conditions at 37 °C. black-square, GDP; open circle , GMP; black-triangle 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (alpha  and beta  isomers, Compounds 6 and 4).

GDP was present as a contamination in the commercial GTP used for certain assays and could be detected by HPLC, but the GDP concentration did not increase during the enzyme reaction. It follows that the enzyme can hydrolyze the alpha /beta - but not the beta /gamma -anhydride bond of GTP without the accompanying release of formate from the imidazole ring of GTP. GMP is generated by the catalytic activity of the enzyme and not by spontaneous hydrolysis.

HPLC analysis monitored by a diode array photometer showed that Compounds 4 and 6 have slightly different absorption maxima at 294 and 291 nm, respectively (Fig. 6). The substrate, GTP, has hardly any absorption at this wavelength. The enzyme-catalyzed reaction can therefore be monitored conveniently via the absorption change at 293 nm (for details see "Experimental Procedures").


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Fig. 6.   Ultraviolet spectra. GTP, ------; Compound 6, alpha -isomer, ····; Compound 4, beta -isomer, - - -. Measurements were performed in the HPLC eluent at pH 7.0.

To study the mechanism of enzyme-catalyzed phosphoanhydride hydrolysis in more detail, GTP was treated with GTP cyclohydrolase II in a buffer containing at least 41% H218O. 31P NMR analysis of the reaction mixture showed satellite signals with up-field shifts of 25 ppb for the N-glycosides and 23 ppb for GMP (Fig. 7). The satellite signals represented ~42% of the total intensity of each respective signal in close accordance with the fraction of H218O in the reaction mixture. On the other hand, the 31P NMR signal of pyrophosphate did not show a heavy isotope satellite. It follows that the enzyme-catalyzed hydrolysis reaction contributes solvent oxygen exclusively to the organic products and not to pyrophosphate.


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Fig. 7.   31P NMR signals of GTP cyclohydrolase II products obtained in buffer containing H218O. For details see "Experimental Procedures."

Steady-state kinetic analysis (data not shown) showed significant deviation from Michaelis-Menten behavior. The data could be best approximated by a cooperative model with a Hill coefficient of 1.33 ± 0.24 (Table IV).

                              
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Table IV
Kinetic properties of wild-type GTP Cyclohydrolase II of E. coli

Foor and Brown (5) have reported that GTP cyclohydrolase II is inhibited by pyrophosphate, but the details had not been studied. We found that pyrophosphate acts as a competitive inhibitor with a KI of 24 ± 7 µM. Orthophosphate acts as a partial uncompetitive inhibitor at low concentrations. At high phosphate concentrations, the type of inhibition changed to a competitive mode (data not shown) (Table IV).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Niederwieser and co-workers (13) have observed two different ultraviolet-absorbing compounds by HPLC analysis of GTP cyclohydrolase II reaction mixtures, but the structures of these compounds were not determined. A more detailed analysis using HPLC and NMR spectroscopy revealed two major products and one minor product. The two major products could be identified as N-ribosyl monophosphates and were assigned as the alpha - and beta -anomers of 2,5-diamino-6-ribosylamino-3(4H)-pyrimidinone 5'-monophosphate (Compounds 6 and 4, Fig. 4). The minor product was identified as GMP.

The origin of the alpha -anomer is attributed to spontaneous isomerization of the beta -anomer produced by enzyme catalysis. Earlier, we reported on the spontaneous anomerization of Compound 2 (Fig. 1), which serves as an intermediate in the reaction catalyzed by GTP cyclohydrolase I (9). The ribose side chains of the formamide derivative 2 and the aminopyrimidine derivative 4 afford similar 13C NMR spectra.

Kinetic analysis of the enzyme-catalyzed reaction by HPLC and 31P NMR spectroscopy indicates that the formation of the alpha -isomer is delayed by comparison with that of the beta -isomer (data not shown). In summary, these results indicate that the alpha -isomer is an artifactual species obtained by spontaneous isomerization.

On the other hand, the data show that the formation of GMP at a low rate is a genuine enzyme-catalyzed reaction because no spontaneous formation of GDP was found to occur under the reaction conditions. This is well in line with data by Kobayashi et al. (18), who showed that the 5'-triphosphates of 8-oxo-7,8-dihydro-2'-deoxyguanosine and 8-oxo-7,8-dihydroguanosine can be converted to the respective monophosphates by GTP cyclohydrolase II, although the enzyme is unable to open the imidazole ring of the structurally modified guanine residues of these nucleotides.

We proposed earlier that the initial step in the reaction catalyzed by GTP cyclohydrolase II could involve the attack of the triphosphate moiety of GTP (Compound 1) by a nucleophilic amino acid side chain affording a phosphoguanosyl derivative of the enzyme (Compound 7) and inorganic pyrophosphate (19) (Fig. 8). The covalently bound nucleotide could then undergo ring opening leading to Compound 8, and the reaction could be terminated by hydrolytic cleavage of the phosphodiester motif. Alternatively, cleavage of the phosphodiester bond without preliminary ring opening should yield GMP (Compound 9) as the final product.


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Fig. 8.   Hypothetical reaction mechanism for GTP cyclohydrolase II.

The proposed mechanism involving covalent catalysis with the formation of a phosphodiester requires the incorporation of 18O from solvent into each of the products, i.e. the 5-aminopyrimidine derivatives 4 and 6 as well as GMP (Compound 9), which was confirmed experimentally. Thus, the experimental data are compatible with the formation of a phosphoguanosyl/enzyme intermediate, but they are not sufficient to prove it.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, European Community Grant ERB FMRX CT98-0204, and the Hans Fischer-Gesellschaft.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: European Molecular Biology Laboratory, 6 rue Jules Horowitz, F-38000 Grenoble, France.

§ Present address: Icon Genetics AG, Blumenstrasse 16, D-85354 Freising-Weihenstephan, Germany.

To whom correspondence should be addressed. Tel.: 49-89-289-13360; Fax: 49-89-289-13363; E-mail: adelbert.bacher@ch.tum.de.

Published, JBC Papers in Press, April 11, 2001, DOI 10.1074/jbc.M100752200

    ABBREVIATIONS

The abbreviation used is: HMQC, heteronuclear multiple quantum coherence.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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