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From the
Received for publication, July 10, 2002, and in revised form, August 19, 2002
The hypothetical protein predicted by the open
reading frame MJ0055 of Methanococcus jannaschii was
expressed in a recombinant Escherichia coli strain under
the control of a synthetic gene optimized for translation in an
eubacterial host. The recombinant protein catalyzes the formation of
the riboflavin precursor 3,4-dihydroxy-2-butanone 4-phosphate from
ribulose 5-phosphate at a rate of 174 nmol mg Flavocoenzymes derived from vitamin B2 (riboflavin)
act as indispensable redox cofactors in all cells (1). They have also been shown to serve a variety of other functions such as DNA
photorepair (2), light sensing (3, 4), and bioluminescence (5-9).
The biosynthesis of riboflavin has been studied in considerable detail
in eubacteria and fungi (Fig. 1) (for
review see Refs. 1 and 10-13). The pyrimidine moiety and the ribityl
side chain of the vitamin are derived from GTP (compound 1),
which is converted to
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5-phosphate, by a series of three enzyme-catalyzed reactions (14-19). The dephosphorylation of this intermediate by a hitherto unknown enzyme
affords
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (compound 2), which serves as substrate for
6,7-dimethyl-8-ribityllumazine synthase (20-22). The second substrate
of that enzyme, 3,4-dihydroxybutanone 4-phosphate (compound
4), is obtained from ribulose 5-phosphate (compound
3) by an unusual rearrangement involving the release of
carbon atom 4 as formate, which is catalyzed by
3,4-dihydroxy-2-butanone 4-phosphate synthase (22-24). Compounds
2 and 4 are condensed by
6,7-dimethyl-8-ribityllumazine synthase under the formation of the
green fluorescent 6,7-dimethyl-8-ribityllumazine (compound
5), phosphate, and water (22, 25). The lumazine 5 subsequently undergoes a dismutation, affording riboflavin (6) and the biosynthetic intermediate 2, which is recycled in the pathway (26-28). In summary, the formation of one equivalent of riboflavin requires one equivalent of compound
1 and two equivalents of compound 3.
The biosynthesis of riboflavin in Archaea has not been studied in
detail. An in vivo study with Methanobacterium
thermoautotrophicum using
[U-13C2]acetate and
[1-13C1]pyruvate as tracers indicated that
the xylene ring of the vitamin is assembled from two 4-carbon units
(29). It was also shown that compound 2 serves as an
intermediate in the biosynthetic pathways of riboflavin as well as the
5-deazaflavin (compound 7) derivative, coenzyme
F420 (30).
Only one enzyme from the archaeal riboflavin pathway, i.e.
riboflavin synthase from M. thermoautotrophicum, has been
reported (31). This paper describes 3,4-dihydroxybutanone-4-phosphate synthase from the Archaeon, Methanococcus jannaschii.
Materials--
5-Nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione
was synthesized by previously published procedures (32, 33).
5-Amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione was freshly prepared by catalytic hydrogenation of
5-nitroso-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (34). [U-13C6]Glucose was purchased from
Isotech (Miamisburg, FL) and was converted to
[U-13C5]ribulose 5-phosphate as described
earlier (24). Recombinant 6,7-dimethyl-8-ribityllumazine synthase of
Escherichia coli was prepared as described previously (35).
Restriction enzymes were from New England Biolabs (Frankfurt am Main,
Germany). T4 DNA ligase was from Invitrogen. Oligonucleotides
were synthesized by Interactiva (Ulm, Germany). DNA fragments were
purified with QiaQuick Gel Extraction Kit from Qiagen (Hilden,
Germany). Taq polymerase was from Eurogentec (Seraign,
Belgium) and from Finnzyme (Epsoo, Finland). Molecular weight standards
were supplied by Sigma.
Strains and Plasmids--
Bacterial strains and plasmids
used in this study are summarized in Table
I.
Restriction Enzyme Digestion of DNA--
DNA was digested at
37 °C with restriction enzymes in reaction buffers specified by the
supplier. The treated DNA was analyzed by horizontal electrophoresis in
0.8-3% agarose gels.
Estimation of Protein Concentration--
Protein concentration
was estimated by a dye binding assay (36) or photometrically
( Construction of Expression Plasmids Used in This
Study--
Ligation mixtures were transformed into E. coli
XL1-Blue cells (37). Transformants were selected on LB solid medium
supplemented with ampicillin (150 mg/l). The plasmids were reisolated
and analyzed by restriction analysis and by DNA sequencing. In all
expression plasmids, the genes coding for ribB and for
mutated ribB of M. jannaschii are under the
control of a T5 promoter and a lac operator.
Gene Synthesis and Construction of an Expression Plasmid--
A
synthetic 109-bp oligonucleotide was prepared by PCR with the
overlapping oligonucleotides MJ-MUT-1 and MJ-MUT-2. In a sequence of
eight PCR amplifications, the oligonucleotides listed in Table II were used pairwise starting with
MJ-MUT-3 and MJ-MUT-4 for the elongation of each prior amplificate. The
final PCR product (651 bp) was digested with EcoRI and
BamHI and was ligated into the plasmid pNCO113 (18, 38),
which had been treated with the same restriction enzymes. The resulting
plasmid designated pNCO-MJ-MUT-wild type was transformed into E. coli XL1-Blue cells.
Site-directed Mutagenesis--
Mutations were performed by PCR
using synthetic oligonucleotides and pNCO-MJ-MUT-wild type as template.
All of the sequences were deposited in the GenBankTM
sequence data base (Table III).
DNA Sequencing--
Sequencing was performed using the Sanger
dideoxy chain termination method (39). Plasmid DNA was isolated from
cultures (5 ml) of XL1-Blue strains grown overnight in LB medium
containing ampicillin (170 mg/l) using QIAprep spin columns from Qiagen.
SDS-Polyacrylamide Gel Electrophoresis--
Sodium dodecyl
sulfate polyacrylamide gel electrophoresis was performed by previously
published procedures (40).
Polyacrylamide Gel Electrophoresis--
Non-denaturating
polyacrylamide gels contained 200 mM phosphate, pH 7.2, 4%
acrylamide, 0.11% bisacrylamide, and 0.1% tetramethylethylenediamine. Polymerization was started by the addition of 0.05% ammonium peroxodisulfate.
Protein Sequencing--
Sequence determination was
performed by the automated Edman method using a 471 A Protein Sequencer
(PerkinElmer Life Sciences).
Purification of 3,4-Dihydroxy-2-butanone-4-phosphate
Synthase--
Recombinant E. coli strains
XL1-pNCO-MJ-MUT (wild type or mutated) were grown in LB
medium containing ampicillin (170 mg liter
Frozen cell mass (5 g) was thawed in 40 ml of 30 mM
potassium phosphate, pH 7.0. The suspension was cooled on ice and
exposed to 60 pulses of a Branson-Sonifier B-12A (Branson Sonic Power Company, Danbury, CT) set to level 5. The suspension was centrifuged (Sorvall SS34 rotor, 15,000 rpm, 15 min, 4 °C). Ammonium sulfate was
added to the supernatant to a final concentration of 2 M. After centrifugation (15,000 rpm, 15 min, 4 °C), the protein
solution was placed on top of a phenyl-Sepharose SP column (40 ml,
Amersham Biosciences), which had been equilibrated with 30 mM potassium phosphate, pH 7.0, containing 1 M
ammonium sulfate. The column was developed with 30 mM
potassium phosphate, pH 7.0. Fractions were combined, concentrated by
ultrafiltration (Ultrafree-15 centrifugal filter device, Millipore,
Eschborn, Germany), and placed on top of a hydroxy apatite column
(40 ml, Amersham Biosciences), which had been equilibrated with 20 mM potassium phosphate, pH 7.0. The column was developed
with a linear gradient of 0-1 M potassium phosphate.
Fractions were combined and concentrated by ultrafiltration.
NMR Assay of 3,4-Dihydroxy-2-butanone-4-phosphate Synthase
Activity--
Assay mixtures contained 100 mM Tris
hydrochloride, pH 7.7, 10 mM MgCl2, and 10 mM [U-13C5]ribulose 5-phosphate
in 10% D2O. The reaction was initiated by the addition of
protein and was monitored by 13C NMR spectroscopy at
37 °C.
Photometric Assay of 3,4-Dihydroxy-2-butanone-4-phosphate
Synthase Activity--
Assay mixtures contained 100 mM
Tris hydrochloride, pH 7.5, 10 mM MgCl2, 10 mM ribose 5-phosphate, 100 µg of
6,7-dimethyl-8-ribityllumazine synthase of E. coli, and 5 units of pentose phosphate isomerase in a total volume of 200 µl. The
mixtures were incubated for 5 min at 37 °C to generate ribulose
5-phosphate. A mixture (200 µl) containing 0.75 mM
freshly prepared
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione and protein was added. The solution was incubated at 37 °C, and absorbance was monitored at 410 nm. An absorbance coefficient of 12,100 M Electrospray Mass Spectroscopy--
Experiments were performed
as described elsewhere (41).
Analytical Ultracentrifugation--
Sedimentation equilibrium
experiments were performed with an analytical ultracentrifuge Optima
XL-I from Beckman Instruments equipped with absorbance optics. Aluminum
double sector cells equipped with quartz windows were used throughout.
Protein concentration was monitored photometrically at 280 nm. Protein
samples were dialyzed against 50 mM potassium phosphate, pH
7.0. The partial specific volume was estimated from the amino acid
composition, affording a value of 0.732 ml g NMR Spectroscopy A hypothetical protein specified by the open reading frame MJ0055
of M. jannaschii shows similarity to
3,4-dihydroxy-2-butanone-4-phosphate synthase of E. coli.
Because open reading frame MJ0055 contained numerous codons, which are
poorly translated in E. coli, we designed a synthetic gene
that was optimized for the codon usage of E. coli.
Approximately 21% (49 of 228) of the codons were replaced, and 10 singular restriction sites spaced at intervals of 10-200 bp were
introduced. The DNA sequence was assembled from 16 synthetic oligonucleotides by a sequence of eight PCR steps as described under
"Experimental Procedures." The synthetic gene was ligated into the
plasmid pNCO113 where it was under the control of a T5 promoter and
lac operator. The sequence of the synthetic gene was
verified by DNA sequencing in both directions and has been deposited in
the GenBankTM data base (accession number AF490554). It was
transcribed efficiently in a recombinant E. coli strain,
affording ~30% of cellular protein.
The recombinant protein obtained by heterologous expression of the
synthetic gene was purified by the chromatographic procedures described
"Experimental Procedures" and was obtained in apparently pure form
as judged by sodium dodecyl sulfate polyacrylamide electrophoresis (Fig. 2).
Biosynthesis of Riboflavin in Archaea Studies on the Mechanism of
3,4-Dihydroxy-2-butanone-4-phosphate Synthase of Methanococcus
jannaschii*
§,,,
,,,, and
Institut für Organische Chemie und
Biochemie, Technische Universität München,
Lichtenbergstrasse 4, D-85747 Garching, Germany, Department
of Protein Crystallography, Max-Planck-Institute of Biochemistry,
Am Klopferspitz 18a, D-82512 Martinsried, Germany, and
¶ Forschungsinstitut für Molekulare Pharmakologie,
Robert-Rössle-Strasse 10, D-13125 Berlin, Germany
ABSTRACT
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1
min1 at 37 °C. The homodimeric 51.6-kDa protein
requires divalent metal ions, preferentially magnesium, for activity.
The reaction involves an intramolecular skeletal rearrangement
as shown by 13C NMR spectroscopy using
[U-13C5]ribulose 5-phosphate as substrate. A
cluster of charged amino acid residues comprising arginine 25, glutamates 26 and 28, and aspartates 21 and 30 is essential for
catalytic activity. Histidine 164 and glutamate 185 were also shown to
be essential for catalytic activity.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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View larger version (14K):
[in a new window]
Fig. 1.
Biosynthesis of riboflavin and
8-hydroxy-5-deazariboflavin.
EXPERIMENTAL PROCEDURES
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Bacterial strains and plasmids used in this study
280 nm = 10600 M1cm1).
Oligonucleotides used for the construction of the synthetic ribB gene
Catalytic properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase
of M. jannaschii
1) at
37 °C. After incubation for 18 h, the cells were harvested by
centrifugation (Sorvall GS3 rotor, 5,000 rpm, 15 min, 4 °C). They were washed twice with 0.9% NaCl and frozen at 
20 °C.
1 cm1 at 410 nm for
6,7-dimethyl-8-ribityllumazine was used for calculation.
1 (42).
--
13C NMR spectra were
recorded at 125.6 MHz using Avance 500 spectrometer from Bruker
Instruments (Karlsruhe, Germany).
RESULTS
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View larger version (77K):
[in a new window]
Fig. 2.
Purification of recombinant
3,4-dihydroxy-2-butanone 4-phosphate synthase from M. jannaschii as shown by SDS-PAGE. Lane
1, SDS-PAGE size markers; lane 2, soluble
fraction of cell extract; lane 3, protein fraction
after precipitation with ammonium sulfate; lane
4, protein fraction after phenyl-Sepharose SP chromatography;
and lane 5, protein fraction after hydroxyapatite
chromatography.
The N-terminal sequence of the recombinant protein was verified by partial Edman degradation affording the sequence MNNVEKAIEALKKGE. A relative mass of 25,799 Da was observed by electrospray mass spectrometry in good agreement with the calculated mass of 25,796 Da.
Sedimentation equilibrium centrifugation of the recombinant M. jannaschii protein afforded a molecular mass of 54 kDa, which indicates a homodimer structure in parallel with the homodimer structure of the E. coli enzyme that has been established by analytical ultracentrifugation and by x-ray crystallography (43, 44).
The reaction catalyzed by the recombinant protein was observed by
13C NMR spectrometry in real time using
[U-13C5]ribulose 5-phosphate as substrate.
The singlet at 170.9 ppm reflects the formate obtained by fragmentation
of the substrate. The other four signals are complex multiplets because
of 13C13C and 13C31P
coupling. Specifically, the doublet of doublets at 25.7 ppm reflects
the methyl group of the enzyme product 3,4-dihydroxy-2-butanone 4-phosphate, which is coupled to the position 2 carbonyl group with a
coupling constant of 40.9 Hz and C-3 with a coupling constant of 13.1 Hz. The carbonyl group of 4 resonates at 211.7 ppm and
appears as a pseudotriplet because of coupling to C-1 and C-3. The
signal of C-3 is a complex multiplet reflecting
13C13C coupling to C-2 and C-4 with coupling
constants of 41 and 40 Hz, to C-1 with a coupling constant of 13.1 Hz,
and to phosphorus with a coupling constant of 7.5 Hz. C-4 of the
product also appears as a multiplet because of coupling to C-3 and to
phosphorus. The 13C spectrum shown in Fig.
3A unequivocally identifies
the enzyme product as 3,4-dihydroxy-2-butanone 4-phosphate. The NMR
data are summarized in Table IV.
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The reaction product obtained from unlabeled ribulose 5-phosphate by the catalytic action of the enzyme is shown in Fig. 3C for comparison. The signals of C-3 and C-4 show 13C31P coupling. The use of the 13C-labeled substrate results in an ~90-fold sensitivity enhancement for the detection of formate, which was crucial for the detection of residual catalytic activity in certain mutant proteins described below.
The spectrum in Fig. 3B was obtained in an enzyme experiment
using a mixture of [U-13C5]ribulose
5-phosphate and unlabeled ribulose 5-phosphate proffered at a ratio of
1:25. The spectrum of the product represents a superposition of the
spectra in Fig. 3, A and C. This is specifically
demonstrated for C-3 in Fig. 4 by the
help of spectral simulation, which accounts rigorously for all observed
lines.
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The enzyme-catalyzed reaction involves breaking of the bonds connecting C-3 and C-5 to C-4 and the formation of a novel bond between C-3 and C-5 of the substrate. Intermolecular recombination of fragments from labeled and unlabeled substrate would be conducive to partially 13C-labeled products, more specifically, to [1,2,3-13C3]compound 4 and [4-13C1]compound 4 as shown in Fig. 4, which shows simulated spectra for C-3 of different isotopomers. The 13C spectrum of compound 4 obtained from a mixture of [U-13C5]ribulose 5-phosphate and unlabeled ribulose 5-phosphate is virtually identical with the superposition of spectra for compound 4 obtained from unlabeled ribulose 5-phosphate and [U-13C5]ribulose 5-phosphate. It follows that the reaction proceeds by a strictly intramolecular mechanism.
Because it is known that both enantiomers of 3,4-dihydroxy-2-butanone
4-phosphate can serve as substrates for 6,7-dimethyl-8-ribityllumazine synthase of Bacillus subtilis (25), we determined
the configuration of the product of M. jannaschii
3,4-dihydroxy-2-butanone-4-phosphate synthase. The CD spectrum shown in
Fig. 5 is that expected for the
L-isomer (23). Hence, the M. jannaschii protein
converts ribulose 5-phosphate into a mixture of formate and
L-3,4-dihydroxy-2-butanone 4-phosphate. It follows that
archaea and eubacteria use the same intermediate as precursor for the
xylene ring of riboflavin.
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Sequence comparison showed that 17 polar amino acids were absolutely
conserved in putative orthologs of the
3,4-dihydroxybutanone-4-phosphate synthase from microorganisms and
plants. Most notably, a short stretch of charged amino acids extending
from position 21 to 30 showed a high degree of overall conservation
(Fig. 6).
|
The hypothetical reaction mechanism (Fig.
7) suggests a crucial role for acid/base
catalysis, and polar ligands are probably involved in the interaction
of the protein with the essential divalent metal ion (Mg2+
or Mn2+). Preliminary NMR studies on enzyme-substrate
complexes had also suggested that the conserved amino acid residues
Thr-112, Thr-115, Asp-118, Arg-119, and Thr-122 interact with the
substrate (45). The x-ray structure of
3,4-dihydroxy-2-butanone-4-phosphate synthase of E. coli in
complex with glycerol, which is believed to bind at the active site as
a fortuitous substrate analog, is also in line with the hypothesis that
the loop comprising the conserved cluster of acidic residues is
directly involved in catalysis (46).
|
The synthetic gene specifying 3,4-dihydroxy-2-butanone 4-phosphate of M. jannaschii could be mutagenized conveniently using various experimental techniques. A total of 33 mutant genes could be expressed efficiently in E. coli. The recombinant proteins were isolated as described under "Experimental Procedures," and their steady-state kinetic parameters were initially determined by a coupled photometric assay using 6,7-dimethyl-8-ribityllumazine synthase as reporter enzyme (Table III). The replacement of glutamate 26, 28, or 185, aspartate 21 or 30, histidine 164, or arginine 25 afforded proteins with relative activities in most cases of <0.7%. Mutants obtained by the replacement of arginine 119 or 161, threonine 165, or glutamate 166 retained significant enzyme activity, but their Km values were increased ~10-fold.
The sensitivity of the photometric assay monitoring the formation of 6,7-dimethyl-8-ribityllumazine is limited by the stability of 3,4-dihydroxy-2-butanone 4-phosphate and 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione. The relatively large Km and the relatively low catalytic rate of the auxiliary enzyme, 6,7-dimethyl-8-ribityllumazine synthase, limit the accuracy of the assay still further, particularly in the case of mutants with low residual activity.
Formate, the second product of 3,4-dihydroxy-2-butanone-4-phosphate
synthase, has the advantage of virtually unlimited stability. For this
reason, we measured the enzyme-catalyzed formation of formate by
13C NMR spectroscopy in real time. To increase the
sensitivity of NMR detection, we used
[U-13C5]ribulose 5-phosphate as substrate.
With the wild type enzyme, this assay recorded an activity of 174 nmol
mg1 min1 as compared with 148 nmol
mg1 min1 determined with the photometric
assay. Energies of activation were 61 and 55 kJ mol1,
respectively, as calculated from the Arrhenius plot (290-330 K) for
the NMR and the photometric assay. The two assays also gave similar
values with mutant proteins, which display relatively high activities
(Table III).
Mutant proteins that had little or no enzyme activity according to the
photometric assay were reanalyzed by the NMR assay. To maximize the
sensitivity, the assay mixture contained protein in the millimolar
range. Operating thus under single turnover conditions, the detection
limit was 40 pmol mg1 min1. That value is
equivalent to one molecule of product formed per subunit during a
period of 16 h. Even at that level of assay sensitivity, the
formation of formate could not be detected with following mutants:
D21E, D21N, E26D, E26Q, E28D, D30E, H164N, and E185D.
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The data reported in this paper show that the formation of the riboflavin precursor, 3,4-dihydroxy-2-butanone 4-phosphate, from ribulose 5-phosphate proceeds similarly in eubacteria, fungi, plants, and archaea. An intramolecular rearrangement precedes the elimination of formate.
The synthetic gene for the expression of the M. jannaschii enzyme enabled the production of the protein in high yield in E. coli. Because the synthetic gene comprises a closely spaced set of unique restriction sites, it is also ideally suited for mutagenesis by the replacement of short cassettes. This enabled the rapid construction of a large set of mutants addressing all conserved polar amino acid residues, which may be essential for catalysis.
The published assay of 3,4-dihydroxy-2-butanone 4-phosphate activity (47) uses an auxiliary enzyme, 6,7-dimethyl-8-ribityllumazine synthase, to monitor 3,4-dihydroxybutanone 4-phosphate. This assay is suitable for samples with a relatively high catalytic activity but becomes error-prone in the case of mutant proteins with minimum activity.
A novel assay reported in this paper detects the formate formed by 3,4-dihydroxy-2-butanone 4-phosphate by 13C NMR spectroscopy. Formate has the advantage of virtually unlimited stability under the experimental conditions. To enhance the sensitivity of the method, we used [U-13C5]ribulose 5-phosphate as substrate that can be easily obtained from [U-13C6]glucose (24). Using this assay, relative activities of 0.02% as compared with the wild type could be detected.
3,4-Dihydroxy-2-butanone-4-phosphate synthases have a characteristic motif of polar amino acids extending from residue 21 to 30 in case of the M. jannaschii enzyme (Fig. 6). Replacement of aspartate 21 or 30 or glutamate 26 or 28 reduces the enzyme activity in most cases to levels of <0.02% as compared with the wild type enzyme. Outside the patch of charged amino acids, the replacement of threonine 112, histidine 164, or glutamate 185 resulted in the reduction of enzyme activity by at least three orders of magnitude.
The hypothetical reaction sequence in Fig. 7 suggests a large number of
proton transfer reactions (around a dozen). Therefore, it is not
surprising that charged amino acids could play a major role in such a
complex reaction trajectory.
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ACKNOWLEDGEMENT |
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We thank A. Werner for help with the preparation of the paper.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF490541-AF490573 and AF516684.
§ To whom correspondence should be addressed. Tel.: 49-89-289-13336; Fax: 49-89-289-13363; E-mail: markus.fischer@ch.tum.de.
Published, JBC Papers in Press, August 27, 2002, DOI 10.1074/jbc.M206863200
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