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
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ABSTRACT |
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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- 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.
-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
-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
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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.
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EXPERIMENTAL PROCEDURES |
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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--ribosylamino-4(3H)-pyrimidinone
5'-phosphate (Compound 4) was estimated photometrically
using an absorption coefficient of
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 min1.
The effluent was monitored photometrically with a diode array photometer.
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RESULTS |
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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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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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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 /
- but not the
/
-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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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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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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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).
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DISCUSSION |
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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 - and
-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 -anomer is attributed to spontaneous isomerization
of the
-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
-isomer is delayed by comparison with that of the
-isomer (data
not shown). In summary, these results indicate that the
-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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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.
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FOOTNOTES |
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* 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.
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
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ABBREVIATIONS |
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The abbreviation used is: HMQC, heteronuclear multiple quantum coherence.
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