From the Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747, Garching, Germany and the § Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907
Received for publication, September 29, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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Conserved amino acid residues of riboflavin
synthase from Escherichia coli were modified by
site-directed mutagenesis. Replacement or deletion of phenylalanine 2 afforded catalytically inactive proteins. S41A and H102Q mutants had
substantially reduced reaction velocities. Replacements of various
other conserved polar residues had little impact on catalytic activity.
19F NMR protein perturbation experiments using a
fluorinated intermediate analog suggest that the N-terminal sequence
motif MFTG is part of one of the substrate-binding sites of the protein.
Riboflavin synthase catalyzes a mechanistically complex
dismutation of 6,7-dimethyl-8-ribityllumazine (Compound 1) affording riboflavin (Compound 3) and
5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione (Compound 4) (Fig. 1) (1-3).
The enzyme is a potential target for the chemotherapy of infections by
Gram-negative bacteria which are unable to absorb riboflavin from the
environment due to the absence of a transport system and are therefore
absolutely dependent on its endogenous synthesis. Specifically, it has
been shown that riboflavin-deficient mutants of Escherichia
coli and Salmonella typhimurium require riboflavin
concentrations well above 10 mg/liter of culture medium (7, 8). Similar
levels of the vitamin are required for the growth of
riboflavin-deficient mutants of the yeasts Saccharomyces
cerevisiae and Candida guilliermondii (9-11).
Inhibitors of riboflavin biosynthesis may therefore also qualify as
potential agents for the therapy of yeast infections, which are
becoming progressively more important in immunocompromised patients.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Hypothetical reaction mechanism of riboflavin
synthase based on the data by Plaut, Wood, and co-workers
(3-6).
The riboflavin synthases of eubacteria and yeasts are homotrimeric proteins with an approximate subunit mass of 24 kDa. The sequence similarity between the N-terminal and C-terminal part of the enzyme suggested that each subunit folds into two domains with similar folding topology (12). In line with this hypothesis, preliminary crystallographic analysis of riboflavin synthase from E. coli provided evidence for pseudo d3 symmetry of the homotrimeric molecule (13).
The reaction mechanism of the enzyme is incompletely understood despite the efforts of several research groups extending over a period of 4 decades (for review, see Refs. 6, and 14). The dismutation reaction involves the transfer of a 4-carbon unit between the two identical substrate molecules and requires the simultaneous presence of two 6,7-dimethyl-8-ribityllumazine molecules at the active site of the enzyme (15-17). This is well in line with the hypothesis of two topologically similar folding domains and with the experimentally observed binding of two substrate molecules per enzyme subunit.
Recently, the two-domain hypothesis received considerable support by
the recombinant expression of N-terminal and C-terminal segments of
riboflavin synthase from E. coli. Both recombinant proteins
were shown to bind the intermediate
analog, Compound 5, but not its diastereomer, Compound
6 (Fig.
2)1
(19). Moreover, the N-terminal domain could bind riboflavin and
6,7-dimethyl-8-ribityllumazine with high affinity. Surprisingly, the
N-terminal domain was found to form a homodimer in contrast to the
heterotrimeric riboflavin synthase.
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The three-dimensional structure of riboflavin synthase is still unknown
despite considerable efforts. Therefore, we decided to use systematic
mutation analysis to identify catalytically relevant amino acid residues.
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EXPERIMENTAL PROCEDURES |
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Materials-- 6,7-Bis(trifluoromethyl)-8-ribityllumazine hydrate (Compound 5) and 6,7-dimethyl-8-ribityllumazine were prepared by published procedures (20, 21).
Restriction enzymes were from New England Biolabs (Schwalbach,
Germany), Amersham Pharmacia Biotech (Freiburg, Germany), or from Life
Technologies, Inc. (Karlsruhe, Germany). T4 DNA ligase was from Life
Technologies, Inc. (Karlsruhe, Germany). Oligonucleotides were
custom-synthesized by Life Technologies, Inc. (Karlsruhe, Germany) or
MWG Biotech (Ebersberg, Germany). Nucleobond AX20 columns for plasmid
DNA purification were from Macherey and Nagel (Düren, Germany).
DNA fragments were purified with the QIAquick Gel Extraction Kit or
QIAquick PCR2 Purification
Kit from Qiagen (Hilden, Germany). Casein hydrolysate and yeast extract
were from Life Technologies, Inc. (Karlsruhe, Germany), isopropyl
-D-thiogalactopyranoside was from Biomol (Hamburg,
Germany), Q-Sepharose Fast Flow and phenyl-Sepharose CL-4B were from
Amersham Pharmacia Biotech (Freiburg, Germany).
Construction of a ribC Hyperexpression Strain-- The ribC gene coding for riboflavin synthase of E. coli was amplified by PCR using the plasmid pERS (22) as template and oligonucleotides 1 and 2 as primers (Table I). The amplificate was purified, digested with XbaI and PstI, and ligated into plasmid pT7-7 (23), which had been digested with the same restriction enzymes. The resulting plasmid was designated pT7-RS.
Bacterial Culture--
Recombinant E. coli strains
were grown in LB medium at 37 °C in shaking flasks to an optical
density of 0.7. Isopropyl--D-thiogalactopyranoside was
added to a concentration of 0.5 mM, and incubation was
continued at 37 °C for 4 h. The cells were harvested by
centrifugation and stored at
75 °C.
Site-directed Mutagenesis-- A procedure modified after Marini et al. (24) was used for site-directed mutagenesis. PCR was performed with Vent DNA Polymerase (New England Biolabs, Schwalbach, Germany) to minimize the error rate. Plasmid pERS was used as template. The internal mismatch primers are shown in the Table I.
The general scheme of mutagenic PCR involved two rounds of amplification cycles using one mismatch and two flanking primers (primers 1 and 2, Table I). During the first round, 5 amplification cycles were carried out with the respective mismatch primer and with one of the flanking primers. Then the second flanking primer was added and the reaction was continued for an 10 additional cycles. The amplificate was subjected to agarose gel electrophoresis, digested with XbaI and PstI, purified using the QIAquick PCR Purification Kit, and ligated into plasmid pT7-7 that had been digested with the same restriction enzymes. The ligation mixture was transformed into E. coli XL1-blue cells (Stratagene, Heidelberg, Germany). Mutant clones were identified by the presence of the respective new restriction site. All plasmid constructs were sequenced by the automated dideoxynucleotide method (Sanger) using a 377 Prism DNA sequencer from Applied Biosystems (Weiterstadt, Germany).
Protein Purification--
Purification procedures were performed
at 4 °C, unless otherwise stated. Frozen cell mass (5 g) was thawed
in 25 ml of 50 mM Tris hydrochloride, pH 7.2, containing
0.5 mM EDTA and 0.5 mM dithiothreitol
(buffer A). The suspension was subjected to ultrasonic treatment and
was then centrifuged. The supernatant was dialyzed against 10 volumes
of buffer A and centrifuged. The supernatant was passed through a
column of Q-Sepharose Fast Flow (2 × 18 cm) pre-equilibrated with
buffer A (flow rate, 1 ml min1). The column was washed
with 100 ml of buffer A and developed with a linear gradient of 0-0.5
M NaCl in buffer A (total volume, 280 ml). Riboflavin
synthase was eluted from 200 to 240 ml. The enzyme fraction was brought
to a concentration of 1 M
(NH4)2SO4 by the slow addition of
an equal volume of 2 M
(NH4)2SO4 in buffer A. The solution
was passed through a column of phenyl-Sepharose CL-4B (1.5 × 12 cm) pre-equilibrated with 1 M
(NH4)2SO4 in buffer A (flow rate, 1 ml min
1). The column was washed with 40 ml of 1 M (NH4)2SO4 in buffer A
and developed with a linear gradient of 1.0-0 M
(NH4)2SO4 in buffer A (total
volume, 120 ml). The enzyme was eluted from 80 to 95 ml. Fractions were
combined, concentrated by ultrafiltration, and dialyzed against 20 mM sodium/potassium phosphate, pH 6.9, containing 0.2 mM EDTA and 0.5 mM dithiothreitol (buffer B).
The dialyzed solution was stored at
75 °C. According to
SDS
polyacrylamide gel electrophoresis (25), the protein samples
contained less than 5% impurities. The oligomeric structure of mutant
proteins was determined using a 6% polyacrylamide nondenaturing gel
electrophoresis system as previously described (26). The protein
concentration was determined photometrically (
280 = 47, 700 M
1 cm
1).
Monitoring of Riboflavin Formation--
Reaction rates were
measured using a stopped-flow module SFM-4 and Diode Array Spectrometer
(Bio-Logic, Claix, France) at 20 °C. All mixing solutions were
prepared in buffer B. The reaction was initiated by rapid mixing of
equal volumes of 8 µM enzyme solution and 25-160
µM 6,7-dimethyl-8-ribityllumazine solution. The reference
absorbance spectrum of 4 µM protein solution in reaction
buffer was automatically subtracted from the experimental data.
Absorbance spectra were collected and analyzed using Spectralys software from J&M Analytische Meß- und Regeltechnik GmbH (Aalen, Germany). Riboflavin concentration was determined using an extinction coefficient 470 = 10,300 M
1
cm
1.
Equilibrium Dialysis--
Equilibrium dialysis experiments were
performed at 24 °C in buffer B using Dianorm microdialysis cells
(Bachofer, Reutlingen, Germany) and Visking dialysis tubes (Medicell,
London, United Kingdom). The protein concentration was 17 µM. The total ligand concentration was between 7.5 µM and 1.1 mM. The dialysis cells were
allowed to equilibrate for 4 h under slow rotation. Control experiments had shown that the concentrations of Compound 5 in the two parts of a microdialysis cell had reached equilibrium after
2 h of incubation. Ligand concentration was determined
photometrically (340 = 7,943 M
1 cm
1).
Protein Perturbation Experiments--
Samples contained 20 mM sodium/potassium phosphate, pH 6.9, 0.2 mM
EDTA, 0.5 mM dithiothreitol, 10% D2O and
protein (10.535.0 mg ml
1). Aliquots (5-30 µl) of
6,7-bis(trifluoromethyl)-8-ribityllumazine hydrate (epimer A) were
added. After each addition, 19F NMR spectra were recorded
at 338 MHz using an AM 360 NMR spectrometer from Bruker Instruments
(Karlsruhe, Germany) at 24 °C. Experimental parameters were as
follows: pulse angle, 30° (2 µs); repetition rate, 1.6 s; 32 K
data set; 800 to 3000 scans. Chemical shifts were referenced to an
external standard containing sodium trifluoroacetate, pH 7.0. The
concentrations of bound and free ligands were calculated from
19F NMR signal integrals.
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RESULTS |
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Construction of Riboflavin Synthase Mutants-- Plaut, Wood, and co-workers (4, 15, 27) had proposed a hypothetical mechanism for riboflavin synthase involving the initial addition of a nucleophile to the lumazine molecule serving as donor of the 4-C unit which is transferred between the two substrate molecules in the enzyme-catalyzed reaction (Fig. 1). More specifically, this hypothetical nucleophile could be either a water molecule or a polar amino acid residue. To check this hypothesis, we decided to mutate polar amino acid residues in search of the hypothetical nucleophile thought to be involved in covalent catalysis.
Sequence comparison of all known or presumed riboflavin synthases of the fungal/eubacterial type showed that the similarity between the proteins from different species was relatively low (data not shown). More specifically, the enzymes from E. coli and the closely related Hemeophilus influenzae share 63% identical amino acid residues. Otherwise, the fraction of identical amino acids was in the range of 29 to 38%.
The sequence comparison revealed 11 polar amino acid residues that
were absolutely conserved (Fig. 3).
Moreover, several other amino acids (Ser41,
Asn45, Thr50, Thr67,
Thr71, Asn83, Tyr133,
Lys137, and Asp143) were rarely replaced. All
these residues were selected for mutagenesis.
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In light of the relatively low overall conservation of the riboflavin synthase sequence, it was most notable that all enzymes invariably had the N-terminal sequence MFXG. A functional role of this sequence segment appeared likely, and it was therefore included in the mutagenesis study.
Site-specific mutagenesis was performed by PCR and generated mutant genes were checked for sequence accuracy by restriction analysis of novel restriction sites and by full-length sequence analysis. Five mutated genes (C48S, T50R, T67R, T148R, and T165R) could not be expressed in recombinant E. coli strains. It was tentatively assumed that the mutated amino acid residues affected the folding and overall conformation rather than the active site of the enzyme, and the respective mutant genes were not studied in more detail. Four mutant genes (Y133A, D143G, D143N, and N181G) (Table I) afforded soluble proteins that were too unstable to be purified. One mutant gene (T3R) could be expressed to a level around 3% of cellular protein in recombinant E. coli host strains. All other mutant genes listed in Table I could be expressed to a level of ~20% of cellular protein and were purified to a level of at least 95%. The migration properties of all mutants as well as the wild type enzyme, in gel electrophoresis under nondenaturing conditions were very similar (data not shown).
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Riboflavin Formation--
The catalytic activity of the mutant
proteins was studied by single turnover experiments using a
stopped-flow photometer. The respective mutant protein and the
substrate, 6,7-dimethyl-8-ribityllumazine, were rapidly mixed at a
molar ratio of 6:1 to 20:1 and the absorbency at 470 nm was recorded.
At this wavelength, the absorbency contribution of
6,7-dimethyl-8-ribityllumazine is low, and the recorded signal is
dominated by the concentration of riboflavin formed. The maximal rate
of riboflavin formation (Vmax) in the case of
the wild type enzyme was 21 nmol mg1 min
1
(Table II), which is equivalent to a
turnover number of 0.5 per min and subunit.
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A substantial number of absolutely or highly conserved, polar amino acid residues could be exchanged without significant impact on the catalytic rate. Thus, the replacement of residues Thr3, Asn45, Glu66, Thr71, Asn83, Glu85, His97, Lys137, Ser146, and Glu183 decreased the reaction velocity by less than 10-fold (Table II).
The replacement of serine 41 by alanine was accompanied by a
substantial reduction in catalytic capacity.
Vmax decreased to 3 pmol mg1
min
1. Similarly, the replacement of histidine 102 by
glutamine afforded a mutant protein with a decreased
Vmax of 83 pmol mg
1
min
1.
The replacement of the absolutely conserved phenylalanine 2 by alanine
afforded soluble protein that had no detectable catalytic activity. No
riboflavin whatsoever could be observed even after long-term incubation
with substrate. Similarly, the deletion of phenylalanine 2 resulted in
the complete loss of catalytic activity. The more conservative
replacement of phenylalanine by tyrosine decreased
Vmax of the enzyme 50-fold, to 0.42 nmol
mg1 min
1.
Protein Perturbation Experiments-- In the absence of protein structural data, we decided to perform protein perturbation studies to characterize the interaction of the mutant proteins with a ligand that binds to the catalytic site of riboflavin synthase. The fluorinated Compounds 5 and 6 have been used extensively in ligand binding studies with riboflavin synthase and lumazine synthase of Bacillus subtilis (17, 20, 28, 29). Fluorine substitution favors the covalent hydration of the pteridine ring system to such an extent that the diasteromeric Compounds 5 and 6 are not subject to epimerization. The covalent hydrate structure mimics the hypothetical intermediate, Compound 2, in the reaction mechanism proposed by Plaut, Wood, and co-workers (Fig. 1) (4, 15, 27).
Riboflavin synthase of B. subtilis has been shown to bind Compound 5 at a molar ratio of 1:1 per subunit (17, 20). Binding of Compound 5 to the enzyme was accompanied by downfield shifts of the 19F NMR signals of both trifluoromethyl groups. Compound 6 (epimer B) could not be bound even at high concentration (20).
A titration experiment performed with E. coli riboflavin
synthase and Compound 5 is shown in Fig.
4. Aliquots of the ligand were added, and
19F NMR spectra were recorded after each addition. At the
early stages of the titration experiment (Fig. 4, a and
b), the 19F NMR signals of the free ligand are
very low in intensity, and the spectra are dominated by the signals of
the enzyme-bound ligand. With progressive saturation of the enzyme's
binding sites, the signals of the free ligand increase. The position 7 trifluoromethyl group of the enzyme-bound ligand appears as a
relatively broad signal with a line width of 180 Hz which is 1.4 ppm
downfield shifted by comparison with the signal of the free ligand. The position 6 trifluoromethyl group of the enzyme-bound ligand affords a
more complex signal pattern. At low ligand concentrations (Fig. 4a), a relatively intense signal with a line width of 160 Hz
is observed at 14.2 ppm and a signal of lower intensity with a line width of 90 Hz at 15.7 ppm. The latter signal increases with
progressive addition of ligand, thus suggesting that the enzyme has two
different types of binding sites whose affinities to Compound
5 differ significantly (Table
III).
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The quantitative evaluation of the signal integrals afforded the
nonlinear Scatchard plot shown in Fig.
5A. Besides the experimental values obtained from the NMR titration, the plot shows experimental values obtained by equilibrium dialysis. The data obtained by the two
different experimental methods fit a model with ~3.2 high affinity
sites (KD = 2.0 µM) and 2.5 low
affinity sites (KD = 41 µM) per enzyme
molecule. Incidentally, it should be noted that the separate evaluation
of the NMR signal integrals affords a KD value of 30 µM for the low affinity site (Fig. 5B).
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These data are well in line with our recent finding that the recombinant N-terminal and C-terminal domains of E. coli riboflavin synthase can both bind Compound 5 (Fig. 4, Table III).1 The comparison between the spectra of the wild type enzyme and the domains suggests tentatively that the ligand signal at 15.7 ppm is associated with the N-terminal domain of the wild type enzyme, whereas the signal at 14.2 ppm is associated with the C-terminal domain.
Titration experiments with Compound 5 were performed with all mutants listed in Table II. In most cases, the spectra were qualitatively similar to that obtained with the wild type enzyme. KD values obtained from 19F NMR titration differ from that of the wild type enzyme by less than a factor of 10 (Table II) and the number of ligand molecules bound per protein molecule (trimer) varied from 5.2 to 6.3 (relative standard error, 6-15%). The few exceptional cases are discussed in detail below.
An NMR titration experiment with the catalytically inactive F2
mutant with a deletion of phenylalanine 2 is shown in Fig. 6. The position 7 trifluoromethyl group
of the bound ligand affords two signals with a distance of 0.9 ppm,
respectively (Table III). On the other hand, the signals of the
position 6 trifluoromethyl group are located very close to each other
with a distance of 0.4 ppm (Fig. 6). The signal pattern suggests that
the mutant retains the capacity to bind one ligand molecule each to the
N-terminal and C-terminal domain. The signals of the ligand molecule
assumed to represent the ligand bound to the N terminus are shifted
upfield (by 1.7 ppm for the 6 trifluoromethyl group and 1.4 ppm for
the position 7 trifluoromethyl group) by comparison with the wild type
enzyme (Table III).
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Titration experiments with the F2 mutant indicate that the different
binding sites bind the ligand with different affinity (Table III). The
low affinity site is characterized by a dissociation constant of 57 µM. The dissociation constant for the high affinity site
cannot be measured accurately for experimental reasons (the NMR signals
of the free ligand are too small to be integrated in the early
titration steps; the value of the dissociation constant is estimated to
be less than 1 µM).
The F2A mutant is very similar to the F2 mutant. It is catalytically
inactive, and it modulates the NMR spectrum of Compound 5 in
the same way as the deletion mutant (Table III). However, the
conservative replacement of phenylalanine 2 by tyrosine affords a
catalytically active protein (Table II) whose interactions with Compound 5 are similar to that of the wild type protein.
Apart from the phenylalanine 2 modifications, only a small number of
mutations analyzed in this study were conducive to substantial modification of the interaction with Compound 5 (Table III,
Fig. 7). In two cases (K137A and S146G),
the catalytic activity was reduced by approximately 1 order of
magnitude, whereas the E183G mutant had almost normal catalytic
activity. In all three mutants, two signals are clearly resolved for
the position 7 CF3 group of the enzyme bound ligand. In the
case of S146G mutant, the signal of the position 6 CF3
group assumed to represent the ligand in association with the
C-terminal domain is shifted upfield by comparison with the wild type
enzyme. In the K137A and E183G mutants, there is tentative evidence for
three enzyme bound species giving rise to additional signals for the
position 6 CF3 group.
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Comparison of the NMR chemical shift values shown in Table III
indicates that, except of Glu183, replacements of other
amino acids in N- or C-teminal halves of riboflavin synthase affected
exclusively (Lys137 and Ser146) or largely
(Phe2) the NMR pattern afforded by the ligand molecule
bound to the same half of the protein. The fact that all mutations
listed in Table III have a visible effect on the appearance of the 6- and 7-trifluoromethyl groups of the ligand, relative to the wild type enzyme, suggests that residues 2, 137, 146, and 183 may be in close
proximity to the ligand and to each other.
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DISCUSSION |
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The reaction catalyzed by riboflavin synthase involves the cleavage of two CN bonds and the formation of two CC bonds. It appears obvious that the reaction trajectory must involve a complex series of intermediates. Surprisingly, however, the reaction can proceed without enzyme catalysis under relatively mild conditions in aqueous solution (30). Notably, the catalyzed and the uncatalyzed reactions share the same regiospecificity (4, 15, 18).
The hypothetical reaction mechanisms suggested by Wood, Plaut, and co-workers (3-6) implicate the addition of a nucleophile to one of the substrate molecules as an early reaction step. In the uncatalyzed reaction, a water molecule could serve this purpose. Either a water molecule or a polar amino acid side chain could serve as a nucleophile in the enzyme-catalyzed reaction.
The protein perturbation data provide evidence for the existence of two different types of binding sites whose affinities for Compound 5 differ by approximately 1 order of magnitude. Evidence for two different types of binding sites had been obtained earlier by studies with riboflavin synthase from B. subtilis and 6-trifluoromethyl-7-oxo-8-ribityllumazine (16, 28), but the 19F NMR spectra produced by Compound 5 in this case not only differed substantially from those shown in Fig. 4, but also gave the molar ratio of 1:1 of bound ligand per subunit, indicating that only one type of binding site could bind the ligand. E. coli and B. subtilis proteins share only 33% identical amino acids (data not shown), so the structural difference between the ligand-binding sites of these two enzymes that leads to the different NMR signal pattern seems plausible.
We have shown recently that the N-terminal and C-terminal parts of the riboflavin synthase of E. coli can be expressed as recombinant proteins.1 Specifically, the N-terminal domain was shown to form a relatively stable, soluble homodimer whereas the full-length riboflavin synthase forms a homotrimer. The recombinant C-terminal domain has comparatively low stability and could not be obtained in pure form. Both recombinant domains were shown to bind Compound 5 by protein perturbation experiments similar to those reported in the present study. A comparison of the chemical shift values indicates that binding of Compound 5 to the N-terminal domain is associated with a relatively large downfield shift of the position 6 trifluoromethyl signal whereas the binding to the C-terminal domain has a more pronounced effect on the signals from the position 7 trifluoromethyl group.
The replacement of serine 41 or histidine 102 reduced the catalytic activity of the enzyme by several orders of magnitude. The mutations did not decrease substantially the affinity of the enzyme for the putative intermediate analog, Compound 5. Moreover, the mutations appeared to have little impact on the environment of enzyme-bound Compound 5 as gleaned by 19F NMR spectrometry. If either of these amino acids were involved in covalent catalysis, the residual activity of the mutant proteins could be due to a water molecule replacing the amino acid nucleophile. Whereas the data are insufficient to prove a direct catalytic role of serine 41 or histidine 102, they rule out covalent catalysis by any of the other polar amino acid residues addressed in the present mutagenesis study.
Whereas this study was primarily focused on conserved polar amino acid
residues, the inclusion of the lipophilic N terminus appeared mandatory
in light of the absolute conservation of the terminal MFXG
motif. Deletion or nonconservative replacement of phenylalanine 2 afforded completely inactive proteins. Although these mutants could
still bind two lumazine-type molecules, the chemical shifts of the
ligand bound at the putative N-terminal-binding site were modulated
significantly. This finding suggests that the N terminus of the
polypeptide forms an essential part of the active site.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft, the Fonds der Chemischen Industrie, the Hans Fischer Gesellschaft, the Alexander von Humboldt-Stiftung (to B. I.), and National Institutes of Health Grant GM51469.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.
On leave of absence from the Institute for Biophysics,
Krasnoyarsk, Russia.
¶ To whom correspondence should be addressed: Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Lichtenbergstr. 4, D-85747 Garching, Germany. Tel.: 49-89-2891-3360. Fax: 49-89-2891-3363; E-mail: adelbert.bacher@ch.tum.de.
Published, JBC Papers in Press, January 18, 2001, DOI 10.1074/jbc.M008931200
1 S. Eberhardt, N. Zingler, K. Kemter, M. Cushman, and A. Bacher, submitted for publication.
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ABBREVIATIONS |
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The abbreviation used is: PCR, polymerase chain reaction.
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REFERENCES |
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