From the Departments of Applied Biological Chemistry
and Biotechnology, Graduate School of Agricultural and Life
Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the ¶ Biotechnology Research Center, University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the ** Department of
Biophysics and Biochemistry, Graduate School of Science, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and the
§§ Department of Microbiology and Immunology,
University of British Columbia, 6174 University Boulevard, Vancouver,
British Columbia V6T 1Z3, Canada
Received for publication, March 21, 2000, and in revised form, September 25, 2000
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ABSTRACT |
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The crystal structure of a major
oxygen-insensitive nitroreductase (NfsA) from Escherichia
coli has been solved by the molecular replacement method at
1.7-Å resolution. This enzyme is a homodimeric flavoprotein with one
FMN cofactor per monomer and catalyzes reduction of nitrocompounds
using NADPH. The structure exhibits an Nitroaromatic compounds including nitrofurans, nitropyrenes, and
nitrobenzenes have been used as antimicrobial agents, food additives
and raw materials in several industrial processes (1-5), and as a
result are distributed widely around the environment. Many of these
compounds are toxic, mutagenic, or carcinogenic (6-8). It is believed
that enzymatic transformation is needed for nitroaromatic compounds to
show these serious effects (9, 10). The reduction of a nitro group of a
parent nitrocompound is a key step of this process (11, 12). Enzymes,
which catalyze the reduction of nitrocompounds using a reduced pyridine
nucleotide, are termed nitroreductases and are distinguished by their
sensitivity of activity to oxygen (9, 10).
The oxygen-sensitive enzymes can catalyze nitroreduction only under
anaerobic conditions. A nitro-anion radical formed by a one-electron
transfer is immediately reoxidized in the presence of oxygen to a
parent nitrocompound and superoxide (13, 14). In this futile cycle,
reducing equivalents are consumed without the progress of
nitroreduction and nitrocompounds perform as a catalyst to reduce
oxygen. On the other hand, the oxygen-insensitive enzymes catalyze an
obligatory two-electron reduction. A nitro group of a parent
nitrocompound is reduced by a series of two-electron transfers, through
nitroso and hydroxylamine intermediates, and finally to an amino group
(13). The hydroxylamine intermediate arising from the four-electron
transfer in total is found to be toxic, carcinogenic, or mutagenic.
Three proteins with oxygen-insensitive nitroreductase activity in
Escherichia coli have been identified (15).
NfsA1 is the major component,
while NfsB and NfsC are minor components. NfsA and NfsB have been well
studied relative to NfsC. NfsA and NfsB have similar enzymatic
property, although NfsA has only 7% identity with NfsB on the amino
acid sequence alignment. Both NfsA and NfsB are flavoenzymes with FMN
as the prosthetic group and catalyze the reduction of nitrocompounds by
Ping Pong Bi Bi kinetics (16, 17). Counterparts of NfsA and NfsB, found
in luminescent bacteria (16, 17), are flavin reductase (FRP) of
Vibrio harveyi (18) and flavin reductase (FRase I) of
Vibrio fischeri (19), respectively. Enzyme that resembles
FRP in the amino acid sequence alignment is also found in
Bacillus subtilus and is called NfrA1 (20). A comparison of
crystal structures solved previously (21-24) indicates that FRP has
structural similarity with FRase I and an NADH oxidase (NOX) of
Thermus thermophilus (24). Thus, NfsA and NfsB seem to
belong to a protein family defined as the nitro/flavin reductase
family, comprised of the NfsA/FRP and the NfsB/FRase I subfamilies.
Interestingly, there is a clear difference in the substrate specificity
between NfsA and FRP, although NfsA shares ~51% amino acid sequence
identity with FRP. Both enzymes utilize NADPH as an electron donor;
however, NfsA cannot accommodate FMN as an electron acceptor (16) and
FRP can reduce FMN in addition to nitrocompounds (20). A single amino
acid substitution in NfsA results in a higher relative activity with
regard to FMN reduction than that of FRP (25). Thus, the determination
of NfsA crystal structure and comparison with the previously determined
FRP crystal structure will improve knowledge of the catalytic reaction
and the substrate recognition.
We report here the crystal structure of NfsA from E. coli,
and discuss the substrate binding in comparison with FRP. Furthermore, we show that Arg203 found in the NfsA/FRP subfamily is
important for NADPH binding.
Protein Purification and Crystallization--
Recombinant
E. coli NfsA was expressed and purified as described
previously (26). Crystals of NfsA were obtained using the hanging drop
vapor diffusion method, with 22.5% PEG 6000 as a precipitant and
2-15% PEG 400, dioxane or isopropyl alcohol as an additive in 100 mM MES buffer (pH 6.5). Crystals were grown at 5 °C,
from 10-µl drops, which contained 5 µl of 9 mg/ml protein solution
and 5 µl of reservoir solution. Plate-shaped crystals appeared in
about 1 week. The unit cell parameters and space group were determined
to be P1 with a = 52.2 Å,
b = 52.7 Å, c = 53.3 Å, Data Collection--
A typical crystal measured ~0.3 × 0.3 × 0.2 mm. To obtain a high-resolution data set with high
completeness, data sets were collected by using both laboratory and
synchrotron x-ray sources. A data set to 2.3-Å resolution was recorded
from single crystals using a Rigaku imaging plate detector system,
R-AXIS IIc, with double mirror-focused CuK Structure Determination and Refinement--
The FRP molecule
(21) was used as the search model for molecular replacement. The
rotation function was calculated with AMORE (28) and gave a single
solution with a correlation coefficient of 36.9%. To improve the
accuracy of the solution, the resultant structure was subjected to 5 cycles of rigid body refinement using data between 4.0- and 10.0-Å
resolution. The crystallographic R-factor and correlation
coefficient was refined to 43.6 and 43.8%, respectively. To improve
Fc and phases, the following procedure was performed
several times. The resulting Fo, Fc, and phases were subjected to density
modification procedures with solvent flattering, histogram matching,
and averaging using DM (29). The improved Fc and
phases were used to calculate a SIGMAA weighted Fo
Further improvement of the model was accomplished by using alternate
cycles of refinement in the program X-PLOR (32, 33) and CNS (35), and
manual intervention using O. Initially strict noncrystallographic
symmmetry was imposed on the model followed by noncrystallographic
symmetry restraints. Approximately 5% of reflections from a number of
thin shells were selected to calculate the free R-factor,
using SFTOOLS.2 The first
round of refinement consisted of positional refinement and simulated
annealing beginning at 3000 K. Later steps of refinement included the
refinement of grouped or individual temperature factors. Simulated
annealing omit maps were used to check and rebuild the region including
residues 202 to 209 in the model. Water molecules were identified by
searching difference maps using WATPEAK in the CCP4 package (34) and
proline are in the most favored regions of the Ramachandran plot and
the rest of the residues except for Asn179 are in the
additional allowed regions. Asn179 is located in a tight
turn between a Construction and Expression of Mutants--
Mutants were
prepared using the polymerase chain reaction (37) with the Quick Change
Site-directed Mutagenesis Kit (Stratagene). A plasmid (pAJ102)
containing the nfsa gene was constructed previously (38).
Synthetic oligonucleotides containing the desired mutation described were used as mutagenic primers,
5'-TACCTCACCGCTGGCAGCAAT-3' and 5'-ATTGCTGCCAGCGGTGAGGTA-3' for R203A;
5'-AGCAATAATGCCCGGGATACC-3' and 5'-GGTATCCCGGGCATTATTGCT-3' for R208A.
Mutagenesis was confirmed by dideoxy sequencing of mutated genes using
a Shimadzu DNA sequencer, DSQ-1000. The mutant NfsAs were expressed and
purified, yielding a single band on SDS-polyacrylamide gel
electrophoresis according to the methods reported previously (26).
Kinetic Characterization of the Mutants--
Cytosolic fractions
were prepared from overnight cultures of E. coli clones
containing wild-type and mutant plasmid constructs. To measure the
relative activity of wild-type enzyme and two mutants, 100 µM of either NADPH or NADH was used as an electron donor
and 100 µM nitrofurazone was used as an electron acceptor
(Table II). Steady-state kinetic analyses
were carried out by monitoring consumption of NADPH by the decrease in
absorbance at 400 nm ( Description of the NfsA Structure--
NfsA is a homodimeric
globular protein of about 45 Å in diameter that contains one FMN per
monomer as a cofactor (Fig. 1). An NfsA
molecule consists of an
As shown in Fig. 2A, the
2Fo
The dimethylbenzene portion of the isoalloxazine group is stabilized by
hydrophobic interaction with helix F in both monomers (Fig. 1). On the
other hand, the O2A/O4A side is partly exposed to the solvent. The O2A
atom forms a hydrogen bond to the N-
The ribityl group of FMN makes three hydrogen bonds (Fig.
2B): O2* with a water molecule, O3* with the amide nitrogen
atom of Ser39B,3
and O4* with the N Comparison of NfsA with FRP--
A superimposition of NfsA onto
FRP yields an r.m.s.d. for C
There is a subtle, but clear difference around the active site pocket
between NfsA and FRP (Fig. 4). The main differences are localized to a
particular region on the left side of the active site, that contains
residues 192 to 225, including a part of helix I, the following
flexible loop and helix J. This region shows an average r.m.s.
deviation of 1.86 Å in distance. Several residues are exposed to the
active site cleft: Tyr199, Tyr200,
Arg203, His215, Arg218,
Thr219, Lys222, Ser224, and
Arg225 in NfsA, and Tyr199, Tyr200,
Trp212, Glu215, Val216,
Lys219, Leu220, and Arg225 in FRP.
Unconserved residues between NfsA and FRP that form part of the active
site are mainly localized on helix J (Fig. 3B). In addition
to the rigid-body movement of helix J between both enzymes (Fig. 4),
substitutions of amino acids contribute to a more closed active site,
especially around position 222, in NfsA relative to FRP (Fig. 4). This
observation is consistent with the specificity for the second
substrate, in that flavins, more bulky molecules than nitrocompounds,
are good substrates for FRP, but poor substrates for NfsA (16, 40).
Fig. 4 also shows that the conserved residues on the amino acid
alignment between NfsA and FRP are not always conserved in their
structures, as observed at position 199 and 200 in NfsA and FRP.
Mutational Analysis of NfsA--
To get direct information about
the NADPH binding and the catalytic pathway of this enzyme, we tried to
provide the crystal of NfsA complexed with NADPH, NADP+, or
its analogues by the soaking method. However, crystals soaked with 100 mM NADPH cracked. As moderate concentration (10 mM or less) of NADPH was used as an additive, the protein
catalyzed the reduction in the crystal, losing the yellow color from
the oxidized form of the cofactor FMN. The diffraction from this
crystal was quite poor and that seemed to be attributed to the disorder of the protein symmetry in the crystal. In using NADP+ or
its analogues, the meaningful electron density could not be found in
the difference Fourier map. This suggests that NADPH binding influences
the conformation of the loop between helices I and J in the vicinity of
the catalytic center FMN, considered that Gly204 and
Arg208 of the loop are involved in the crystal packing interaction.
The solvent exposed loop region between helices I and J is ~20 Å away from the catalytic center (isoalloxazine ring) of the cofactor FMN
(Fig. 4). However, this loop could be close to the binding site of the
adenosine moiety of NADPH bound to the active site of NfsA in an
extended conformation. This loop has a flexible conformation as shown
by the average B-factor of 25.5 Å2. The crystal
packing may define the loop conformation in the crystal and it is
unclear whether this structure of the loop is the most stabile in solution.
To investigate the role of the flexible loop consisting of residues 202 to 211 in the putative NADPH-binding site, two positively charged amino
acids, Arg203 and Arg208, were chosen for
site-directed mutagenesis (Fig. 3B). These two residues were
considered to be candidates for NADPH binding in the FRP crystal
structure (21). Arg203 is one of the completely conserved
residues in the active site according to the amino acid sequence
alignment among NfsA, NfrA1, and FRP. On the other hand,
Arg208 in NfsA corresponds to Lys208 in FRP and
Arg217 in NfrA1. It is possible that the side chain of
Arg208 recognizes NADPH considering the possibility of
refolding of the flexible loop in solution, although Arg208
is not directed toward the active site in the present structure. As
observed in other NADPH binding enzymes, there are examples where
positively charged residues, such as arginine, lysine, and histidine,
are involved in the recognition of the 2'-phosphate group of NADPH
through a hydrogen bond (41-45). Thus, if
Arg203 or Arg208 interact with NADPH, mutation
of these residues will contribute to a decrease in NADPH binding
affinity to NfsA.
The catalytic activities of whole cell extracts of the mutant and wild
type enzymes were measured using NADPH as an electron donor and
nitrofurazone as an electron acceptor. As compared with the relative
activity of the wild-type enzyme, the R203A mutant showed low activity
(Table II). On the other hand, the relative activity of the R208A
mutant is similar to that of the wild-type enzyme (Table II). To
characterize R203A NfsA further, kinetic parameters were determined.
The Km value for NADPH of R203A NfsA is 33-fold
higher than the Km of the wild-type, whereas the
Km value for nitrofurazone of R203A enzyme is
comparable to that of wild-type (Table
III, data of wild-type enzyme were
obtained and modified from Ref. 16). These findings suggest that R203A
but not R208A is involved in interactions with NADPH, most probably
through the 2'-phosphate.
Comparison of FMN Binding--
The binding conformation of the
cofactor FMN in NfsA is quite similar to that in FRP (21) and several
other flavoproteins (23, 24). Comparing FMN binding of NfsA with that
of FRP reveals highly conserved hydrogen bonds and hydrophobic
interactions that contribute to FMN binding. The main difference
between NfsA and FRP in the flavin environment is the replacement of
His69 in NfsA with Tyr69 in FRP (Fig.
2B). The O The Specificity of the Second Substrate in the NfsA/FRP
Subfamily--
NfsA catalyzes poorly the reduction of flavins (16),
whereas NfrA1 and FRP have broader electron acceptor specificity and catalyze efficiently the reduction of not only nitrocompounds but also
flavins (20). Considering the differences in the active sites between
NfsA and FRP suggests that the specificity for the second substrate
depends partly on the volume of the active site pocket. Indeed, a
previous mutagenesis study indicates that NfsA is able to accommodate
FMN as the second substrate by substituting Glu to Gly at position 99 (25). The side chain of Glu99 is located at the interface
between the two monomers and participates in three hydrogen bonds. The
O Proposed Binding Mechanism of NADPH to NfsA--
To consider a
possible mechanism of NADPH binding, two minimum requirements may be
met. One, that the 2'-phosphate group of NADPH interacts with
Arg203 (see Table II) and, two, that the nicotinamide ring
of NADPH is located near the isoalloxazine ring of FMN for hydride ion transfer between the two rings. In addition, in the crystal structures of NAD(P)+-related oxidoreductases with flavin compounds as
cofactors (46-48), the nicotinamide ring of NADPH is stacked with the
isoalloxazine ring of FMN such that the N1-C4 axis of the nicotinamide
ring is approximately parallel to the N5-N10 axis of the isoalloxazine ring. To best fulfill these conditions, we have tried to build a model
of the NfsA-NADPH complex using the present crystal structure of NfsA
and the conformations of NAD(P)+ observed in protein
crystals (49). The parallel arrangement of the nicotinamide N1-C4 axis
with the isoalloxazine N5-N10 axis could not be satisfied in the
NfsA-NADPH complex model without considerable conformational change of
the NADPH and/or NfsA. Indeed, if the nicotine ring is arranged to
satisfy the stacking and parallel arrangement, steric hindrance of the
ribose connected to the nicotine ring occurs with the ribityl portion
of FMN or the region near Phe42 of NfsA. The parallel
arrangement of the axes of FMN and NADPH, thus, prevents the stacking
of the two rings, and consequently interaction of 2'-phosphate group of
NADPH with Arg203.
Fig. 5 shows the proposed model of
NfsA-NADPH complex, in which the conformation of NADP is taken from
that bound to 6-phosphogluconate dehydrogenase (PDB code 1PGO) (43),
which possesses the most extended conformation among the
NAD(P)+ conformations observed in protein crystal
structures (49). As a result, the N1-C4 axis of NADPH inclines ~60°
relative to the N5-N10 axis of FMN in the model. The nicotinamide
moiety of NADPH interacts with Arg225 and the surrounding
residues Arg133 and Asn134 (Fig. 5). The
negatively charged pyrophosphate is likely to attract the positively
charged Lys167 and Arg15, that are active site
residues conserved between the structures of NfsA and FRP, both of
which use NADPH specifically as an electron donor. If NfsA undergoes a
conformational change by NADPH induced-fit, a complex satisfying the
conditions described above could be formed during the catalytic
reaction in solution. Thus, the present model may need revising to give
a complete picture of the binding mode of NADPH.
Preliminary measured catalytic activities of whole cell extracts of
wild-type, R203A, and R208A enzymes using NADH as an electron donor
showed that quite low, but similar relative activity to one another
(Table II). One possible presumption is that the difference in the
activity shown only when using NADPH results from the difference in the
structure between NADPH and NADH, and also that the 2'-phosphate peculiar to NADPH interacts with the side chain of Arg203.
The observation that R203A mutant has nitroreductase activity when
using NADPH suggests that several amino acids might participate in the
recognition of the 2'-phosphate group. In this proposed model, the
2'-phosphate is located in the vicinity of a hydrophilic cluster formed
by Arg203 and surrounding residues, such as
Asp165, Tyr199, and Tyr200 (Fig.
5). These residues are also conserved, except that Gly171
in NfrA1 and Glu165 in FRP corresponding to
Asp165 in NfsA.
+
-fold, and is comprised
of a central domain and an excursion domain. The overall structure of
NfsA is similar to the NADPH-dependent flavin reductase of
Vibrio harveyi, despite definite difference in the spatial
arrangement of residues around the putative substrate-binding site. On
the basis of the crystal structure of NfsA and its alignment with the
V. harveyi flavin reductase and the
NADPH-dependent nitro/flavin reductase of Bacillus
subtilis, residues Arg203 and Arg208 of
the loop region between helices I and J in the vicinity of the
calalytic center FMN is predicted as a determinant for NADPH binding.
The R203A mutant results in a 33-fold increase in the Km value for NADPH indicating that the side chain
of Arg203 plays a key role in binding NADPH possibly to
interact with the 2'-phosphate group.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
= 75.1°,
= 60.6° and
= 60.5°. On the basis of the
assumption of one dimer per unit cell, the calculated Vm value
is 2.08 Å3/Da. Data set statistics are given in Table
I.
Data collection and refinement statistics for NfsA
radiation from a Rigaku
RU-200R x-ray generator. A cooling device was used to keep the crystal
at 100 K (Rigaku CN2364B2). The cell parameters of NfsA crystals were determined by processing data collected on the R-AXIS IIc. To refine
the crystal structure of NfsA, a data set to 1.7-Å resolution was
collected from a single crystal under cryo-condition using synchrotron
radiation at station BL6A at the Photon Factory at the High Energy
Accelerator Research Organization (Tsukuba, Japan). Data frames were
collected using the Weissenberg camera for macromolecular crystallography, an imaging plate, and a Fuji image reader (BAS2000) and were processed using the data reduction programs DENZO and SCALEPACK (27). To collect the diffraction data efficiently, both
Weissenberg and oscillation photographs were used, due to the
difficulty to align crystals for the Weissenberg method under cryo-cooling conditions.
Fc electron density map (30). The density map
was displayed in the program O (31) and used to rebuild the model manually.
-strand and
-helix. The overall geometry (36) is
good as compared with proteins of the same resolution. R.m.s deviations
of distances and angles are 0.009 Å and 1.745°, respectively.
Crystallographic data and refinement statistics are shown in Table
I.
= 12.96 mM
1
cm
1) (13) in reaction mixtures containing appropriate
amounts of the purified enzymes in 50 mM Tris-HCl buffer,
pH 7.0, at 23 °C. To determine the Km values for
both NADPH and nitrofurazone, the initial rates of the reactions were
observed at varying concentrations of NADPH in the range of 5 to 200 µM for each of the various nitrofurazone concentrations
in the range of 5-200 µM. NADPH in the reaction mixture
was not saturating to determine the Km of R203A mutant for NADPH. It was difficult to obtain accurate kinetic data when
using more than 200 µM NADPH, since the absorption curves of both substrates overlapped each other near the measured wavelength. An estimate of the Km for NADPH and the value for
Vmax was obtained by extrapolation for this
mutant. The analysis of the data was performed using the program
Cleland (39).
Cytosolic nitroreductase activity of the wild-type and mutant enzymes
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
+
-fold formed by 12
-helices and
five
-strands and is divided into two domains, the central domain
and the excursion domain. The central domain is composed of residues
15-161 and 226-240, and forms an anti-parallel
-sheet structure
comprised of strands 1-4. On one side of the central
-sheet,
helices C, D, and G lie almost vertically relative to the
-sheet,
and on the other side, the sheet is surrounded by helices B and F (Fig.
1). The excursion domain consists of residues 1-14 of one monomer and
residues 162-225 of the other monomer. Residues 1-14 form helix A,
which contacts with helices B and F in the central domain. Residues
162-225 forms a long coil element (residues 162-171) followed by
helix H and strand 5, which is parallel to strand 1 in the central
domain (Fig. 1) and connect helix I through a tight turn at the end of
strand 5. A loop (residues 202-211) and helix J form a large part of
one side of the cleft made between two polypeptide chains of the dimer
(Fig. 1). The loop between residues 202 and 211 has high
B-factor values and forms a flexible loop.
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Fig. 1.
The C trace of
NfsA. Helices are represented with green cylinders and
strands with dark orange plane ribbons. Two FMN
cofactors are drawn in a ball and stick model. The protein is viewed
along the noncrystallographic 2-fold axis. The arrows
labeled 3 and the cylinder labeled
310 indicate strand 3 and 310 helix,
respectively. This figure and following figures were drawn using the
program MOLSCRIPT (51).
Fc electron density
map is of high quality at the interface of both monomers in the NfsA
crystal structure. The re face of the isoalloxazine portion
of cofactor FMN faces the putative substrate-binding site. The average
B-factor for two FMNs is 4.3 Å2, consistent
with FMN being bound tightly through many hydrogen bonds provided by
surrounding main chain and side chain atoms of the peptide chain (Fig.
2B).
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Fig. 2.
The environment of the cofactor FMN in
NfsA. A, the electron density of FMN from a
2Fo Fc map contoured at one
. This figure was prepared using the program O. B, a
stereo diagram of hydrogen bonds network between the cofactor FMN and
NfsA. The enzyme is drawn as a ball and stick model. Each atom is
colored according to atom type. Hydrogen bonds are indicated with
black broken lines. In this and all subsequent figures, the
single letter code for amino acid names is used for enhanced
clarity.
of Arg15 and a
water molecule. The O4A atom forms hydrogen bonds to the amide
nitrogens of Gly130 and Gly131, and the N
-2
atom of Gln67. The N-1 and N-5 atoms, the likely redox site
of FMN, have hydrogen bonds with the N
-2 atom of Arg15
and the amide nitrogen of Gly130, respectively. On the
si face of FMN, the carbonyl oxygen of Tyr128 is
located only 2.9 Å away from the C4A atom of the pyrazine portion of
FMN. This interaction makes the isoalloxazine ring slightly bent even
in the oxidized form as observed in FRP, FRase I, and NOX (21-24).
atom of Lys167. The negative charge
of the phosphate is balanced by the positively charged N
-1 and
N
-2 atoms of Arg169 and the N
-2 atom of
His11, and also forms a hydrogen bond to the amide nitrogen
atom of Ser13. Additional water-bridged hydrogen bonds are
present with the N
-1 and N
-2 atoms of Arg15, the side
chain amide nitrogen atom of Lys167 and the oxygen atom of
His11, to stabilize of the phosphate.
atoms of 0.54 Å for the entire
polypeptide chain (Fig. 3A).
The superimposition of these two structures reveals that the residues that form the active site pocket except for helices I and J superimpose well on each other (Fig. 4). The well
conserved residues are localized on the right side of the active site.
Especially Arg15, Gln67, Arg133,
Lys167, Arg225, Ser40B, and
Ser41B are conserved at the atomic level of the
three-dimensional structure (Fig. 4). Considering the same substrate
specificity with regard to the first substrate between NfsA and FRP in
the Ping Pong Bi Bi catalytic system, some of these conserved and
correspondent residues might be involved in NADPH binding.
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Fig. 3.
Comparison of NfsA with FRP.
A, superimposition of NfsA onto FRP (Protein Data Bank entry
1BKJ). NfsA and FRP are drawn as -carbon traces and are colored in
magenta and green, respectively. FMN cofactors
are represented in a dark orange stick model. The
superimposition was accomplished with the program Clusterid (50).
Residues 201-209 are missing in the FRP coordinates. B,
amino acid sequence alignment of NfsA and FRP. NfrA1 is also aligned.
Identical amino acids are indicated by bold face letters.
Deletions are shown by the symbol,
. Residues whose side chains form
the active site cleft are labeled by an asterisk (*)
above the sequence in NfsA and below the sequence
in FRP, respectively. Residues subjected to mutation are
boxed. Secondary stucture assignments of NfsA and FRP are
shown above and below the amino acid sequences,
respectively.
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Fig. 4.
The active site of the NfsA/FRP
subfamily. Superimposition of the FMN (depicted in dark
orange stick model) of NfsA onto that of FRP. NfsA and FRP are
colored in magenta and green, respectively.
Residues around the active site are drawn. The side chains of
Glu165, Glu215, and Lys219 are
missing from the FRP coordinates. The superimposition was produced
using the program O.
Kinetic parameters of the purified wild-type and mutant enzyme
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
atom of Tyr69 in FRP forms a
hydrogen bond to the O2A atom (21), while a water molecule is located
within a distance capable of making a hydrogen bond to the O2A atom of
the FMN in NfsA. The N-5 nitrogen atom of the FMN is exposed to the
solvent, according to a solvent accessibility calculation using a
1.4-Å probe (Fig. 2B). One of two N-1 atoms in the NfsA
dimer is completely solvent inaccessible, suggesting that the N-5 atom
is the likely candidate for receiving a hydride from the C-4 atom of
NADPH in the NADPH oxidation process.
-1 atom forms a hydrogen bond with the N
atom of
Arg133 on the 310 helix and the O
-2 atom
forms a hydrogen bond with the N
-2 atom of Arg133
and the N
-1 atom of Arg225.
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Fig. 5.
Proposed binding mode of NADPH in the active
site of NfsA. An NADPH molecule derived from the crystal structure
of 6-phosphogluconate dehydrogenase (43) (Protein Data Bank entry 1PGO)
is overlaid on the active site. The putative residues involved in NADPH
binding are drawn in a ball and stick model. Each atom is colored
according to atom type. Chemical bonds of protein and NADPH are colored
in green and those of FMN in yellow. The two
monomers of the molecule are colored in dark orange and
blue.
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ACKNOWLEDGEMENTS |
---|
We thank Professor N. Sakabe and Drs. N. Watanabe, M. Suzuki, and N. Igarashi of the Photon Factory at the High Energy Accelerator Research Organization for data collection. We thank Professor T. Nishino of Nippon Medical School for critical reading of the manuscript. We are grateful to Dr. H. Koike for advice.
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FOOTNOTES |
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* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science and Culture of Japan, the Sakabe project of TARA, University of Tsukuba, performed in part using the Station BL6A at the Photon Factory, High Energy Accelerator Research Organization under the approval of the Photon Factory Program Advisory Committee Proposal number 97G091.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 atomic coordinates and the structure factors (code 1F5V, 1PGO, and 1BKJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: National Food Research Institute, Ministry of Agriculture, Forestry and Fisheries, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8642, Japan.
Present address: Dept. of Physiology, Medical University of
Oita, 1-1 Idaigaoka, Hasama, Oita 879-5503, Japan.
§§ To whom correspondence should be addressed: Dept. of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-5165; Fax: 81-3-5841-8023; E-mail: amtanok@ mail.ecc.u-tokyo.ac.jp.
Published, JBC Papers in Press, October 6, 2000, DOI 10.1074/jbc.M002617200
2 B. Hazes, unpublished results.
3 Residue marked with B is from the other monomer.
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ABBREVIATIONS |
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The abbreviations used are: NfsA, the major oxygen-insensitive NADPH-dependent nitroreductase of Escherichia coli; NfsB, the minor oxygen-insensitive NAD(P)H-dependent nitroreductase of E. coli; FRP, an NADPH-dependent flavin reductase of V. harveyi; NOX, an NADH oxidase of T. thermophilus; NfrA1, an NADPH-dependent nitro/flavin reductase of B. subtilis; FRase I, the major NAD(P)H:FMN oxidoreductase of V. fischeri; r.m.s., root mean square.
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