From the RIKEN Harima Institute/SPring-8,
1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, the
§ Department of Virology, Osaka City University Medical
School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan, and the
¶ Osaka Seiki Women's College, 3-10-62 Aikawa,
Higashiyodogawa-ku, Osaka 533-0007, Japan
Received for publication, November 13, 2002, and in revised form, December 27, 2002
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ABSTRACT |
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Cytochrome P450 isolated from Bacillus
subtilis (P450BS Two bacterial cytochrome P450s isolated from Sphingomonas
paucimobilis and Bacillus subtilis,
P450SP In typical P450 reactions an oxygen atom derived from molecular oxygen
(O2) is inserted into the substrates (7), and the reaction
is referred to as a monooxygenation reaction. Two protons and two
electrons are required in the monooxygenation reaction. The electrons
are supplied from NAD(P)H through mediation by flavoproteins and
iron-sulfur proteins, and the protons are probably delivered from
solvent water to the active site through a specific hydrogen-bonding network (8). In the monooxygenase P450 system,
H2O2 is sometimes used as a surrogate for the
O2/2e Concerning the site specificity of the reaction, the To address these two issues concerning the unique peroxygenases
P450BS Preparation of Recombinant Forms of P450BS Crystallization and Data Collection--
Crystals of
P450BS
Multi-wavelength anomalous diffraction data of the protein crystal
containing iron of the heme were collected at the RIKEN beam line
(BL44B2) station of SPring-8, Hyogo, Japan, with a MAR CCD detector
(15). The crystals were mounted in cryo-loops directly from the mother
liquor and frozen in liquid nitrogen. Data were processed with the
HKL2000 package (16).
Phasing and Refinement of P450BS Overall Fold and Potential Surface--
Fig.
1 shows the crystal structure of ferric
P450BS
Fig. 2, A and B
show the charge distribution on the proximal (heme-Cys side) and the
distal (heme sixth coordination side) surfaces of
P450BS
The features on the surface potential distribution of
P450BS
In sharp contrast, P450BS Substrate Binding Channel of P450BS
As stated above, the substrate fatty acid could be stabilized in
P450BS Site Specificity in Hydroxylation by P450 Peroxygenases--
As
stated in the Introduction, P450SP
Before analyzing the products of the catalytic reaction, the binding of
fatty acids to these mutants was examined in the solution state using
resonance Raman spectroscopic techniques (Fig.
4A). The binding of the
substrate to P450BS
The resonance Raman spectra of the three mutants (F79L, V170F,
F79L/V170F) in the ferric resting and the ferrous CO forms are shown in
Fig. 4, A and B, respectively. In the spectrum of the ferric F79L mutant, the
On the other hand, the F79L/V170F double mutant and the V170F single
mutant exhibited only 10 and 30% of the activity of the WT enzyme,
respectively. The
The mutation study suggested that Val170
directly affects the binding of a fatty acid to P450BS Heme Active Site Structure and Possible Reaction Mechanism of
Peroxygenase P450--
In the heme proximal side of
P450BS
The heme pocket structure of P450BS
One of the most interesting findings in the active site structure of
P450BS
It should be noted here that the configuration of
(Arg242-fatty acid carboxylate-H2O-Fe)
observed in P450BS
One of the most striking and characteristic differences in the
mechanism of P450BS
Lastly, we discuss briefly the role of channel II of
P450BS
Thus far, crystal structures of a number of P450s have been determined,
and it has been found that their basic structures are very similar.
However, the P450 superfamily covers a variety of biological catalytic
reactions with a variety of substrates and reactants. Such functional
diversity in P450s can be attributed to their structural diversity,
which might be related to substrate recognition and functional sites at
the active site. In the present study, the functional diversity of
peroxygenase P450BS; molecular mass, 48 kDa)
catalyzes the hydroxylation of a long-chain fatty acid
(e.g. myristic acid) at the
- and
-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450BS
in the substrate-bound form,
determined at a resolution of 2.1 Å. P450BS
exhibits a
typical P450 fold. The substrate binds to a specific channel in the
enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by
electrostatic interaction of its terminal carboxylate with the
Arg242 guanidium group. These interactions are
responsible for the site specificity of the hydroxylation site in which
the
- and
-positions of the fatty acid come into close proximity
to the heme iron sixth site. The fatty acid carboxylate group interacts
with Arg242 in the same fashion as has been reported for
the active site of chloroperoxidase,
His105-Glu183, which is an acid-base catalyst
in the peroxidation reactions. On the basis of these observations, a
possible mechanism for the hydroxylation reaction catalyzed by
P450BS
is proposed in which the carboxylate of the
bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
1 and
P450BS
, respectively, are heme-containing enzymes that catalyze the hydroxylation reaction of long chain fatty acids (e.g. myristic acid) using hydrogen peroxide
(H2O2) as an oxidant to produce hydroxylated
(-OH) fatty acids (1, 2). In the enzymatic reactions, an oxygen atom
derived from H2O2 is efficiently introduced
into the substrate with a high catalytic turnover (1,000 min
1) (2-4). P450SP
produces the
-OH
fatty acid (100%) as the product, whereas P450BS
produces both the
-OH (60%) and the
-OH (40%) fatty acids (1,
2, 4, 5). The amino acid sequence of the two enzymes shares a 44%
identity (2). Data base investigation has shown that
P450SP
and P450BS
belong to the P450
superfamily and, therefore, they have been given the systematic
nomenclature designations CYP152B1 and CYP152A1, respectively (6).
However, when compared with reactions catalyzed by other P450s, two
characteristic properties in the P450SP
and
P450BS
reactions were found, i.e. the
utilization of H2O2 and the site specificity of
the reaction.
/2H+ system (peroxide shunt
pathway), but the turnover rate in the peroxide shunt pathway is
generally much lower than that in the reduction system-supported
catalytic cycle. In contrast, P450SP
and
P450BS
did not require any reduction system of
O2 for their catalysis. Indeed, ferredoxin,
ferredoxin reductase, and P450 reductase systems do not appear to
function in the P450SP
and P450BS
reactions.2 Both enzymes
specifically require H2O2 for their catalytic
activities in place of O2/2e
/2H+,
and, thus, the peroxide shunt pathway is the main route to hydroxylated fatty acid production. These bacterial P450 enzymes, therefore, should
be appropriately designated as peroxygenase rather than monooxygenase
(1, 2, 4, 9, 10).
- and
-hydroxylation of fatty acids by P450SP
and
P450BS
are very unique compared with monooxygenase P450s
such as P450 from Bacillus megaterium (P450BM3), which are
known to catalyze the hydroxylation at the
-n
(n = 1, 2, 3) sites of fatty acids. In the crystal
structure of the substrate-bound form of P450BM3 it was found that the
substrate (palmitoleic acid) binds to the enzyme through an ionic
interaction of the carboxylate with a phenol group of Tyr51
located on the protein surface and through hydrophobic interactions of
the long alkyl chain with the hydrophobic interior of the protein (11).
Most recently, Haines et al. (12) reported on the crystal structure of P450BM3 in which the carboxylate group of the substrate, N-palmitoylglycine, interacts with the backbone nitrogen
atoms of Gln73 and Ala74 on the surface of the
protein. In any event, such interactions result in the
CH3-terminal (
-position) of the alkyl chain of the fatty
acid being located near the active center, leading to
-1,
-2, and
-3 hydroxylated products. In contrast, P450SP
and
P450BS
give an
- and/or a
-OH fatty acid as the
products of the hydroxylation reaction. Therefore, the
- and
-carbons (-C
H2-C
H2-COOH)
must be located in close proximity to the heme active site. This
suggests that the substrate-enzyme interaction for
P450SP
and P450BS
is entirely different from that for P450BM3. The difference in the site specificity of the
reaction must be due to differences in structures involved in the
substrate recognition between peroxygenase P450s (P450SP
and P450BS
) and monooxygenase P450s.
and P450SP
properties, we
attempted to crystallize the enzymes for crystallographic analyses.
Fortunately, we were successful in obtaining a crystal of
P450BS
(13) and report herein on its crystal structures
determined at 2.1-Å resolution, which provide the structural basis of
understanding the characteristic properties of the peroxygenase
P450BS
described above.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
and
Assay of the Hydroxylation Activities of Myristic Acid--
A
recombinant enzyme of P450BS
was expressed from
Escherichia coli M15 (pREP4) and purified according to
previously reported methods (14). The site-directed mutagenesis for
preparing some mutants of the enzyme, i.e. F79L, V170F, and
F79L/V170F, was achieved using PCR with pQE-30t BS
as a template.
The purification of the mutants was achieved using the same method as
was used for the wild type (WT) enzyme. Assay methods for the myristic
acid hydroxylation activities of the enzymes have been reported
elsewhere (14).
were grown in a sitting drop by the vapor
diffusion method at 20 °C as described previously (13). These
crystals were obtained as follows: 20 mg/ml protein was mixed with an
equal volume of the reservoir solution consisting of 10%(w/v)
polyethylene glycol (PEG) 3350 (Sigma) and 50 mM MES at pH
6.8. The crystal belongs to the trigonal space group
P3221 with cell dimensions a = b = 155.8 Å, c = 111.9 Å. The
asymmetric unit contains three polypeptide chains and three heme molecules.
--
The initial
phases were obtained using SOLVE (17). Three iron sites were located,
and the resulted phases have a figure of merit of 0.22. Density
modification was performed with RESOLVE (18), resulting in a clearly
interpretable electron density map. The P450BS
polypeptide chain was manually built into this map using O (19). The
initial model was submitted to simulated annealing with a slow cool
protocol using the crystallography NMR software (CNS) program (20) with
high-resolution data. Energy minimization and individual temperature
factor refinement was used, alternated by manual fitting into the
density. Bulk solvent and overall anisotropic B-factor corrections were
applied. A non-crystallographic symmetry restraint was used at the
initial stage of the refinement. The final model consists of three
polypeptide chains with residues number 6-416, 295 water molecules,
and three palmitic acids. Using reflections in the resolution range
20-2.1 Å, the R factor is 24.6%, and the free
R factor (21) is 28.0%. The stereochemical quality of the
refined model was verified with the PROCHECK (22) and WHAT CHECK (23)
programs. Refinement statistics are given in Table I. The
obtained coordinates of the P450BS
structure has been
deposited in the Protein Data Bank
(1IZO). The figures were prepared using MOLSCRIPT (24), BOBSCRIPT (25),
RASTER3D (26), O (19), and GRASP (27). The correlation coefficient and
real space R factors of the electron density map were
calculated by O (19).
Phasing and refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
determined at a 2.1 Å resolution. It exhibits
the typical P450 fold (8, 11, 12, 28-36); the molecule is in a
triangular shape with a side 60-Å long and a 30-Å thickness and is
divided into
-rich and
-rich domains. A long I helix runs across
the entire molecule in the heme sixth (distal) side and is locally
distorted at Pro243, very close to the sixth iron
coordination position (see below).
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Fig. 1.
Top (A) and side
(B) views of the overall structure of ferric
cytochrome P450BS . The substrate binding
channel I and II are represented as a shadow. The heme and palmitic
acid molecules are shown as a ball-and-stick model in purple
and pink, respectively. The I helix is colored
blue.
, respectively. In the proximal surface of
P450BS
(Fig. 2A), some negatively charged
(acidic) residues such as Glu113, Glu216,
Glu275, Glu341, Glu343,
Glu358, Glu366, and Glu371 are
characteristically present. On the other hand, in the distal surface of
P450BS
(Fig. 2B), a positively charged
(basic) cluster consisting of Lys17, Arg40,
Lys44, Lys72, Arg73,
Lys76, Arg179, Arg184, and
Arg399 is specifically present.
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Fig. 2.
Electrostatic potential surface of
P450BS . Proximal (A) and distal
(B) sides are shown. The negatively charged region is
represented in red, while the positively charged region is
in blue.
are in sharp contrast to those of other
monooxygenase P450s in which a positively charged cluster is present on
the proximal surface, whereas a negatively charged region is present on
the distal surface (28, 37). It has been generally thought that such a
characteristic distribution of the charged residues on the surface is
highly related to the electron and proton deliveries to the iron site
in P450 monooxygenation reactions. The positively charged region on the
proximal surface of monooxygenase P450s represents a possible direct
interaction site with the negatively charged surface of their redox
partners (reductases) in the electron-transfer step for the
O2 activation (37). Protons are delivered from the
negatively charged distal surface to the iron site through a specific
hydrogen-bonding network (8).
does not require any electrons
and protons for catalytic activity, because it utilizes
H2O2 as an oxidant instead of
O2/2e
/2H+. This enzyme requires
neither a reductase nor a proton delivery system. Therefore, the
differences observed in the surface potential distribution are quite
consistent with the functional differences between
P450BS
and monooxygenase P450s, in particular with regard to the electron and proton transfer steps in the catalytic cycle.
--
In the
structure of P450BS
(Fig. 1), two channels, I (10 Å in
diameter and 23 Å in length) and II (8 Å in diameter and 18 Å in
length), are identified around the B', F, and G helices. Both channels
are open so as to connect the heme active site (protein interior) to
the protein surface (protein exterior). A positively charged cluster,
which is described above, is located near the entry of channel I (Fig.
2B). Channel I is occupied by an organic compound whose
electron density map is illustrated in Fig.
3. The electron density map shows a good
consistency with a fatty acid because it consists of a long chain and a
Y-shaped terminus. The long chain is surrounded by several hydrophobic
side chains, which are shown in Fig. 3, and the length of the chain is
consistent with 15 carbon atoms, i.e.
CH3(CH2)14-. Palmitic acid was
fitted into this density, yielding a correlation coefficient of 0.843 and a real space R factor of 22.2% between the model and a
A-weighted omit Fo
Fc
map. The terminal group of this long chain, probably a
CH3-group, reaches to the surface of the enzyme. On the
other hand, another terminus, i.e. the Y-shaped terminus, is
located very close to the iron sixth (active) site of the enzyme and
interacts with the guanidium group of Arg242 in the I helix
(see Fig. 3). The distance between the Y-shaped group and the
Arg242 guanidium group (Nh2 and
N
atoms) is 2.8 Å, which falls in the range of
electrostatic or hydrogen-bonding interactions. Based on these
observations, we assigned the compound to palmitic acid
(CH3(CH2)14COOH) derived from
E. coli and assigned the channel I to the substrate binding
site of P450BS
. E. coli contains large
amounts of palmitic acid (38).
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Fig. 3.
Substrate binding in P450BS
(stereo view). The SIGMAA (43)-weighted Fo
Fc map (blue) shows the fatty-acid
electron density peak. The contour is drawn at the 2.2-
level. The
refined palmitic acid model
(CH3(CH2)14COO
) gives
a correlation coefficient and real space R-factor of 0.843 and 22.2%, respectively. Hydrophobic side chains surround the long
alkyl chain. The density of the Y shaped terminus corresponds to the
carboxyl group of palmitic acid, which interacts with the guanidium
group (N
2 and N
atom) of
Arg242. The distances between iron atom and atoms in
palmitic acid is 4.9 Å (Fe-O), 5.1 Å (Fe-C
), or 6.4 Å (Fe-C
).
by two interactions from the protein portion, i.e. hydrophobic interactions with the alkyl side chain and
electrostatic interactions with the carboxyl group. In the case of
hydrophobic interactions, the side chains of Leu17,
Leu70, Val74, Leu78,
Phe79, Val170, Phe173,
Ala246, Phe289, and Phe292 are
involved, whereas Arg242 mainly contributes to the
electrostatic interactions (see Fig. 3). We previously reported that
the R242A mutant of P450BS
was devoid of any
peroxygenation activities (14). The loss of enzymatic activity in this
mutation was likely the result of a loss of the electrostatic
interactions between the carboxylate and the 242 group in
P450BS
. As a result of these specific interactions, the
- and
-carbons are positioned near the iron sixth coordination
site; 5.0 Å for Fe-C
and 6.2 Å for
Fe-C
. The configuration of the fatty acids (substrate)
in P450BS
is entirely different from that in P450BM3
(11, 12) in which the
-1,
-2 and
-3 positions of fatty acids
are closest to the heme iron. The effects of the hydrophobic
interactions between the alkyl group and hydrophobic residues on
substrate recognition by peroxygenase P450BS
are
discussed in the next section.
isolated from
S. paucimobilis is, like P450BS
, also a
peroxygenase, and their reaction mechanisms are possibly the same.
However, it is noteworthy that the hydroxylated products are somewhat
different; 100% of the
-OH fatty acid is produced in the case of
P450SP
, whereas 60% of the
-OH and 40% of the
-OH fatty acids are produced in the case of P450BS
.
The difference in this reaction site specificity is indicative of a
difference in the configuration of the bound substrate relative to the
active iron site. Because Arg242 in P450BS
corresponds to Arg241 in P450SP
, the
electrostatic interaction of the fatty acid carboxylate is the same for
the two peroxygenases (2). In comparing amino acid residues involved in
hydrophobic interactions with alkyl chains of substrates (see Fig. 3),
it was found that Phe79 and Val170 in
P450BS
are substituted for Leu78 and
Phe169 in P450SP
, respectively. To address
their importance in determining the hydroxylation site specificity in
peroxygenase P450s, we prepared three mutants of P450BS
in which Phe79 was replaced with leucine (F79L),
Val170 with phenylalanine (V170F), and the double mutant
(F79L/V170F).
produces a subtle change in optical
absorption and the resonance Raman spectra of the ferric resting
enzyme, because the heme iron is in a low spin state in the
substrate-free form and even in the substrate-bound form (5). In the
resonance Raman spectra of the WT enzyme, only one band, at 374 cm
1, was shifted to 369 cm
1 (5 cm
1 downfield shift) on the binding of substrate
(myristic acid) (5). The band is tentatively assigned to the bending
mode of the heme propionate (
propionate), because this
mode gives a band at 377 cm
1 for myoglobin (39). Indeed,
the channel I for substrates in P450BS
is located
adjacent to the heme 7 propionate, as can be seen in Fig. 3, so that
the bound substrate is able to interact with the heme propionate,
probably resulting in a change in the propionate bending mode. In
addition, we observed the Fe-CO stretching band (
Fe-CO)
also changed from 488 to 491 cm
1 (upfield shift by 3 cm
1) when the substrate was added to the ferrous CO form
of the WT enzyme (Fig. 4B). The bound substrate is located
sufficiently close to permit an interaction with the iron-bound CO,
thus changing the character of the Fe-CO bond. Based on these
observations, these two Raman bands can be utilized to monitor the
substrate binding to P450BS
. Indeed, in the Raman
spectra of the R242A mutant, the position of both bands
(
propionate and
Fe-CO) remained unchanged
when substrate was added (data not shown).
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Fig. 4.
Resonance Raman spectra of
P450BS and its mutants. a, WT; b,
F74L; c, V170F; d, F74L/V170F. The spectra
A and B were measured for the enzymes in the
ferric resting and ferrous CO states, respectively, in the presence and
absence of myristic acid (MA).
propionate band shifts from
375 to 371 cm
1 (downfield shift by 4 cm
1)
upon the addition of myristic acid, indicating that the fatty acid
binds to this mutant. However, the
Fe-CO band exhibits a downfield shift from 488 to 487 cm
1. The relative
position of the bound fatty acid to the CO is probably different
between the WT and F79L mutant enzymes. Correspondingly, as shown in
Table II it can be seen that, in the case
of the F79L mutation for P450BS
, although the specific
activity was reduced by half, the
/
ratio changes from 1.4 to
0.2. In the reaction catalyzed by this mutant, the
-OH product is
dominant.
Analyses of product and specific activities of P450BS and
its mutants (F79L, V171F, and F79L/V170F)
/
ratio was unaltered in the case of the V170F
mutation, showing that these mutations are not suitable for the
peroxygenase activity of P450BS
. In the resonance Raman
spectra, the positions of the
propionate bands of both mutants observed at 375 cm
1 hardly changes when the
substrate fatty acid is added, although the position of the
Fe-CO band of the V170F mutant is shifted from 476 to
478 cm
1 (upfield shift by 2 cm
1). The
myristic acid either might bind to the mutant enzymes in a quite
different way from that for the WT enzyme or might not bind to the
enzyme at all.
because this residue locates very close to the substrate binding site,
with the distance between Arg242 and Val170
being 3.8 Å. On the other hand, the Phe79 residue in
P450BS
is one of the important residues that control the
- or
-site specificity of the hydroxylation reaction catalyzed by
peroxygenases P450. In the crystal structure of P450BS
, Phe79 is located 4.7 Å from the alkyl chain of the bound
fatty acid. Unfortunately, how Phe79 affects the position
of the fatty acid relative to the heme iron, is unclear at present.
, a thiolate of Cys363 is coordinated
to the iron as a fifth ligand (dFe-S = 2.3Å). The coordination of the Cys363 thiolate is stabilized through
hydrogen-bonding interactions with its surrounding residues,
i.e. the amide NH of Gln352 and the main chain
NHs of Gly365 and Glu366, as shown in Fig.
5. In monooxygenase P450s, the crystal
structures of which are available, structural characteristics usually
referred to as the "Cys ligand loop" are observed in their fifth
ligand side in which the main chain NHs of three residues next to the ligand Cys are involved in stabilizing the Fe-S bond. In
P450BS
, although the residue next to Cys363
is proline (Pro364), which cannot interact with the
coordinate Cys363 thiolate, the Gln352 amide
NH, in place of Pro364, is involved in this interaction.
Furthermore it was found for P450BS
that three amino
acid residues are inserted before the bound Cys residue in the primary
sequence and that these extra residues form a longer loop, as shown in
Fig. 5. In the alignment of the primary sequence with a number of
P450s, we expected some structural differences around the ligand
Cys363 between monooxygenases and peroxygenases (4), but
the crystallographic analysis unambiguously revealed that their
structural characteristics are essentially the same for their
three-dimensional structures.
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Fig. 5.
The "Cys ligand loop" structure in the
heme proximal side of P450BS (stereo view).
is illustrated in
Fig. 6. At the sixth iron coordination
site a water molecule is present with a 2.0-Å Fe-H2O bond
distance. The heme iron in the ferric resting state is hexacoordinated,
consistent with optical absorption spectra. The ferric iron of
peroxygenase P450BS
is in a ferric low spin state even
in the substrate-bound form. The sixth ligand water also interacts with
the carboxylate oxygen atom of the substrate fatty acid through a
hydrogen bond with a distance of 3.2 Å. The distance of the
Fe-O(carboxylate) is 5.3 Å. Because the oxygen atom is present just
above the heme iron, the Fe-H2O-O(carboxylate) is on the
heme axis, i.e. the angle of the
Fe-H2O-O(carboxylate) is estimated to be 171°. The carboxylate of the substrate fatty acid further interacts with the
guanidium group of Arg242, as stated above (see Figs. 3 and
5). In addition to Arg242, some hydrophilic residues such
as Asn239 and Gln85 are present in the active
site of P450BS
, making for a quite polar heme pocket
compared with the apolar heme pocket of P450BM3. This polar heme
environment of P450BS
could accommodate the polar
carboxylate group of the fatty acid and H2O2.
Arg242 is followed by Pro243 in the I helix so
that the normal hydrogen-bonding pattern of the backbone residues of
the helix is interrupted at this site, and consequently the helix has a
13° kink centered on this interruption (35).
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Fig. 6.
Heme pocket structure of
P450BS (stereo view). Side (A) and top
(B) views of the heme plane are illustrated. The heme
(purple), palmitic acid (gray), and the hydrogen
bonding network of the water molecules (dashed
line) are shown. The carboxyl group of the substrate
interacts with the guanidium group of Arg242
(light blue) in the I helix (green
ribbon). Oxygen and nitrogen atoms are colored
red and blue, respectively. The fifth ligand Cys
is represented in yellow.
is that the positions of Pro243 and
Arg242 correspond to those of the so-called "conserved"
threonine and acidic residues found in most monooxygenases P450. In the
P450 monooxygenation reaction, the alcoholic -OH and the -COOH at these positions are involved in formation of a specific hydrogen bonding network, which plays a crucial role in the delivery of protons during
the catalytic reaction. Some water molecules take part in this proton
delivery system (8). However, because of the substitution of amino acid
groups in these positions and the absence of water molecules, no
hydrogen-bonding network is present at the heme active site of
P450BS
that can connect the active site to the solvent
region. Therefore, the heme active site of P450BS
is
isolated from the solvent water region, as shown in Fig. 6. This
structural characteristic in the heme pocket is in good agreement with
the catalytic function of P450BS
, which does not require
a proton supply during its catalytic reaction.
is apparently similar to that of
(His105-Glu183
carboxylate-H2O-Fe) found in chloroperoxidase (CPO) (40).
On the basis of the CPO structure, a possible mechanism for the
peroxidation reaction has been proposed, in which the carboxylate of
Glu183 acts as an acid-base catalyst for the O-O bond
cleavage of hydrogen peroxide (H2O2), and its
basicity is modulated through hydrogen bonding with the imidazole of
His105 (41). By analogy with the CPO reaction mechanism, we
propose a molecular mechanism of peroxygenation reaction of
P450BS
as follows (see Scheme
1). The substrate-free enzyme of
P450BS
(Scheme 1A) is inactive because its
optical absorption spectrum remained unchanged on the addition of
H2O2 to the enzyme in this state. Therefore,
the substrate-bound form constitutes the active state for this enzyme
(Scheme 1B). The incoming H2O2
displaces the water molecule from the sixth coordination site, and the
carboxylate of the fatty acid (the substrate) abstracts a peroxide
proton (Scheme 1C). The singly ionized peroxide covalently
binds to the iron, forming a short-lived intermediate followed by the
uptake of a proton to the terminal oxygen atom by the fatty acid
carboxylate, leading to the heterolytic cleavage of the O-O bond of the
peroxide (Scheme 1D) to give the iron in the higher
oxidation state (formally porphyrin Fe5+ = O or equivalent
state; Scheme 1E). In a similar manner to His105 in
CPO, Arg242 of P450BS
could modulate the
basicity of the fatty acid carboxylate, which could serve as the
acid-base catalyst for the heterolytic cleavage of
H2O2. The resulting intermediate could hydroxylate the substrate fatty acid at either the
- or
-position, possibly according to a radical rebound mechanism, giving
either the
- or
-OH fatty acid as a product. In the crystal
structure of the ferric enzyme, the
- and
-positions of the
substrate fatty acid are located at a distance of 5.0 and 6.2 Å from
the iron, respectively, apparently far for the hydroxylation reaction. However, it is possible that the substrate could move close to the iron
in other iron states, because NMR studies of P450BM3 revealed that the
position of the substrate is different between the ferric and the
ferrous iron states (42).
View larger version (15K):
[in a new window]
Scheme 1.
Proposed mechanism of the
P450BS peroxygenase reaction.
, compared with that of CPO, is the participation of the bound substrate in O-O bond cleavage. This is a
first example of the substrate-assisted peroxygenation reaction catalyzed by a cytochrome P450 type enzyme. We recently prepared the
R242K mutant of P450BS
, in which Arg242 had
been replaced with lysine. A spectroscopic study suggested that
myristic acid would be able to bind to this mutant (data not shown),
possibly through electrostatic interactions between the carboxylate and
the Lys242
-amino group. However, the mutant exhibited a
quite low peroxygenation activity (14). One possible explanation for
these observations might be a different configuration of the
carboxylate, relative to the heme iron, between the WT and the R242K
mutant enzymes.
, even though no direct evidence is available. In
the channel II, a water chain that connects the heme propionate and the
solvent region was observed (Figs. 1 and 6). This might be a channel
for a water molecule to escape from the active site to the solvent after its generation in the peroxygenation reaction. In addition, hydrogen peroxide might arrive at the heme site from the exterior of
the protein through this channel, because no channel is available in
the substrate-bound enzyme.
can be clearly discussed on the
basis of structural diversity that has been crystallographically determined.
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FOOTNOTES |
---|
* This work was supported in part by grants from the Structural Biology and the Molecular Ensemble Programs in RIKEN (to Y. S.) and Grant-in-aids from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to I. M. and Y. S.).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 1IZO) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/). .
Present address: Division of Protein Design, Graduate School
of Integrated Science, Yokohama City University, 1-7-29 Suehiro, Tsurumi, Yokohama, 230-0045, Japan.
** To whom correspondence should be addressed. E-mail: yshiro@ mailman.riken.go.jp.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M211575200
2 I. Matsunaga, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
P450SP, cytochrome P450 isolated from Sphingomonas
paucimobilis;
P450BS
, cytochrome P450 isolated from
Bacillus subtilis;
WT, wild type;
MES, 4-morpholineethanesulfonic acid;
CPO, chloroperoxidase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Matsunaga, I., Sumimoto, T., Ueda, A., Kusunose, E., and Ichihara, K. (2000) Lipids 35, 365-371[Medline] [Order article via Infotrieve] |
2. | Matsunaga, I., Ueda, A., Fujiwara, N., Sumimoto, T., and Ichihara, K. (1999) Lipids 34, 841-846[Medline] [Order article via Infotrieve] |
3. | Matsunaga, I., Yamada, M., Kusunose, E., Miki, T., and Ichihara, K. (1998) J. Biochem. (Tokyo) 124, 105-110[Abstract] |
4. |
Matsunaga, I.,
Yokotani, N.,
Gotoh, O.,
Kusunose, E.,
Yamada, M.,
and Ichihara, K.
(1997)
J. Biol. Chem.
272,
23592-23596 |
5. | Matsunaga, I., Yamada, A., Lee, D. S., Obayashi, E., Fujiwara, N., Kobayashi, K., Ogura, H., and Shiro, Y. (2002) Biochemistry 41, 1886-1892[CrossRef][Medline] [Order article via Infotrieve] |
6. | Nelson, D. R., Kamataki, T., Waxman, D. J., Guengerich, F. P., Estabrook, R. W., Feyereisen, R., Gonzalez, F. J., Coon, M. J., Gunsalus, I. C., Gotoh, O., Okuda, K., and Nebert, D. W. (1993) DNA Cell Biol. 12, 1-51[Medline] [Order article via Infotrieve] |
7. | Sono, M., Roach, M. P., Coulter, E. D., and Dawson, J. H. (1996) Chem. Rev. 96, 2841-2888[CrossRef][Medline] [Order article via Infotrieve] |
8. |
Schlichting, I.,
Berendzen, J.,
Chu, K.,
Stock, A. M.,
Maves, S. A.,
Benson, D. E.,
Sweet, R. M.,
Ringe, D.,
Petsko, G. A.,
and Sligar, S. G.
(2000)
Science
287,
1615-1622 |
9. | Matsunaga, I., Yamada, M., Kusunose, E., Nishiuchi, Y., Yano, I., and Ichihara, K. (1996) FEBS Lett. 386, 252-254[CrossRef][Medline] [Order article via Infotrieve] |
10. | Matsunaga, I., Kusunose, E., Yano, I., and Ichihara, K. (1994) Biochem. Biophys. Res. Commun. 201, 1554-1560[CrossRef][Medline] [Order article via Infotrieve] |
11. | Li, H., and Poulos, T. L. (1997) Nat. Struct. Biol. 4, 140-146[Medline] [Order article via Infotrieve] |
12. | Haines, D. C., Tomchick, D. R., Machius, M., and Peterson, J. A. (2001) Biochemistry 40, 13456-13465[CrossRef][Medline] [Order article via Infotrieve] |
13. | Lee, D. S., Yamada, A., Matsunaga, I., Ichihara, K., Adachi, S., Park, S. Y., and Shiro, Y. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 687-689[CrossRef][Medline] [Order article via Infotrieve] |
14. | Matsunaga, I., Ueda, A., Sumimoto, T., Ichihara, K., Ayata, M., and Ogura, H. (2001) Arch. Biochem. Biophys. 394, 45-53[CrossRef][Medline] [Order article via Infotrieve] |
15. | Adachi, S., Oguchi, T., Tanida, H., Park, S.-Y., Shimizu, H., Miyatake, H., Kamiya, N., Shiro, Y., Inoue, Y., Ueki, T., and Iizuka, T. (2001) Nucl. Instrum. Methods Phys. Res. A 467-468, 711-714 |
16. | Otwinowsky, A., and Minor, W. (1997) Methods Enzymol. 276, 307-326 |
17. | Terwilliger, T. C., and Berendzen, J. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 849-861[CrossRef][Medline] [Order article via Infotrieve] |
18. | Terwilliger, T. C. (1999) Acta Crystallogr. Sect. D Biol. Crystallogr. 55, 1863-1871[CrossRef][Medline] [Order article via Infotrieve] |
19. | Jones, T. A., Zou, J. Y., Cowan, S. W., and Kjeldgaard, M. (1991) Acta Crystallogr A 47, 110-119[CrossRef][Medline] [Order article via Infotrieve] |
20. | Brunger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve] |
21. | Brunger, A. T. (1992) Nature 355, 472-475[CrossRef] |
22. | Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef] |
23. | Vriend, G. (1990) J. Mol. Graph. 8, 52-56[CrossRef][Medline] [Order article via Infotrieve] |
24. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |
25. | Esnouf, R. M. (1999) Acta Crystallogr. Sect. D Biol. 55, 938-940[CrossRef] |
26. | Merritt, E. A., and Bacon, D. J. (1997) Methods Enzymol. 277, 505-524 |
27. | Nicholls, A., Sharp, K., and Honig, B. (1991) Proteins 11, 281-296[Medline] [Order article via Infotrieve] |
28. | Shimizu, H., Park, S. Y., Shiro, Y., and Adachi, S. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 81-89[CrossRef][Medline] [Order article via Infotrieve] |
29. | Poulos, T. L., Finzel, B. C., and Howard, A. J. (1987) J. Mol. Biol. 195, 687-700[Medline] [Order article via Infotrieve] |
30. | Hasemann, C. A., Ravichandran, K. G., Peterson, J. A., and Deisenhofer, J. (1994) J. Mol. Biol. 236, 1169-1185[Medline] [Order article via Infotrieve] |
31. | Cupp-Vickery, J. R., and Poulos, T. L. (1995) Nat. Struct. Biol. 2, 144-153[Medline] [Order article via Infotrieve] |
32. | Williams, P. A., Cosme, J., Sridhar, V., Johnson, E. F., and McRee, D. E. (2000) Mol. Cell 5, 121-131[Medline] [Order article via Infotrieve] |
33. |
Yano, J. K.,
Koo, L. S.,
Schuller, D. J.,
Li, H.,
Ortiz de Montellano, P. R.,
and Poulos, T. L.
(2000)
J. Biol. Chem.
275,
31086-31092 |
34. | Park, S., Yamane, K., Adachi, S., Shiro, Y., Weiss, K., Maves, S., and Sligar, S. (2002) J. Inorg. Biochem. 91, 491[CrossRef][Medline] [Order article via Infotrieve] |
35. |
Podust, L. M.,
Poulos, T. L.,
and Waterman, M. R.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
3068-3073 |
36. | Mowat, C. G., Leys, D., McLean, K. J., Rivers, S. L., Richmond, A., Munro, A. W., Ortiz Lombardia, M., Alzari, P. M., Reid, G. A., Chapman, S. K., and Walkinshaw, M. D. (2002) Acta Crystallogr. Sect. D Biol. Crystallogr. 58, 704-705[CrossRef][Medline] [Order article via Infotrieve] |
37. | Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., and Deisenhofer, J. (1995) Structure 3, 41-62[Medline] [Order article via Infotrieve] |
38. |
Allen, E. E.,
and Bartlett, D. H.
(2000)
J. Bacteriol.
182,
1264-1271 |
39. | Feis, A., Marzocchi, M. P., Paoli, M., and Smulevich, G. (1994) Biochemistry 33, 4577-4583[Medline] [Order article via Infotrieve] |
40. | Sundaramoorthy, M., Terner, J., and Poulos, T. L. (1995) Structure 3, 1367-1377[Medline] [Order article via Infotrieve] |
41. | Sundaramoorthy, M., Terner, J., and Poulos, T. L. (1998) Chem. Biol. 5, 461-473[Medline] [Order article via Infotrieve] |
42. | Modi, S., Sutcliffe, M. J., Primrose, W. U., Lian, L. Y., and Roberts, G. C. (1996) Nat. Struct. Biol. 3, 414-417[Medline] [Order article via Infotrieve] |
43. | Read, R. J. (1986) Acta Crystallogr. A 42, 140-149[CrossRef] |