Substrate Recognition and Molecular Mechanism of Fatty Acid Hydroxylation by Cytochrome P450 from Bacillus subtilis

CRYSTALLOGRAPHIC, SPECTROSCOPIC, AND MUTATIONAL STUDIES*

Dong-Sun LeeDagger , Akari YamadaDagger , Hiroshi SugimotoDagger , Isamu Matsunaga§, Hisashi Ogura§, Kosuke Ichihara, Shin-ichi AdachiDagger , Sam-Yong ParkDagger ||, and Yoshitsugu ShiroDagger **

From the Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta ; molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha - and beta -positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450BSbeta in the substrate-bound form, determined at a resolution of 2.1 Å. P450BSbeta 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 alpha - and beta -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 P450BSbeta is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Two bacterial cytochrome P450s isolated from Sphingomonas paucimobilis and Bacillus subtilis, P450SPalpha 1 and P450BSbeta , 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). P450SPalpha produces the alpha -OH fatty acid (100%) as the product, whereas P450BSbeta produces both the beta -OH (60%) and the alpha -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 P450SPalpha and P450BSbeta 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 P450SPalpha and P450BSbeta reactions were found, i.e. the utilization of H2O2 and the site specificity of the reaction.

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-/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, P450SPalpha and P450BSbeta 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 P450SPalpha and P450BSbeta 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).

Concerning the site specificity of the reaction, the alpha - and beta -hydroxylation of fatty acids by P450SPalpha and P450BSbeta are very unique compared with monooxygenase P450s such as P450 from Bacillus megaterium (P450BM3), which are known to catalyze the hydroxylation at the omega -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 (omega -position) of the alkyl chain of the fatty acid being located near the active center, leading to omega -1, omega -2, and omega -3 hydroxylated products. In contrast, P450SPalpha and P450BSbeta give an alpha - and/or a beta -OH fatty acid as the products of the hydroxylation reaction. Therefore, the alpha - and beta -carbons (-Cbeta H2-Calpha H2-COOH) must be located in close proximity to the heme active site. This suggests that the substrate-enzyme interaction for P450SPalpha and P450BSbeta 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 (P450SPalpha and P450BSbeta ) and monooxygenase P450s.

To address these two issues concerning the unique peroxygenases P450BSbeta and P450SPalpha properties, we attempted to crystallize the enzymes for crystallographic analyses. Fortunately, we were successful in obtaining a crystal of P450BSbeta (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 P450BSbeta described above.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Preparation of Recombinant Forms of P450BSbeta and Assay of the Hydroxylation Activities of Myristic Acid-- A recombinant enzyme of P450BSbeta 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 BSbeta 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).

Crystallization and Data Collection-- Crystals of P450BSbeta 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.

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 P450BSbeta -- 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 P450BSbeta 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 P450BSbeta 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).

                              
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Table I
Phasing and refinement statistics


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Overall Fold and Potential Surface-- Fig. 1 shows the crystal structure of ferric P450BSbeta 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 alpha -rich and beta -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 P450BSbeta . 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.

Fig. 2, A and B show the charge distribution on the proximal (heme-Cys side) and the distal (heme sixth coordination side) surfaces of P450BSbeta , respectively. In the proximal surface of P450BSbeta (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 P450BSbeta (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 P450BSbeta . Proximal (A) and distal (B) sides are shown. The negatively charged region is represented in red, while the positively charged region is in blue.

The features on the surface potential distribution of P450BSbeta 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).

In sharp contrast, P450BSbeta 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 P450BSbeta and monooxygenase P450s, in particular with regard to the electron and proton transfer steps in the catalytic cycle.

Substrate Binding Channel of P450BSbeta -- In the structure of P450BSbeta (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 sigma 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 Nepsilon 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 P450BSbeta . E. coli contains large amounts of palmitic acid (38).


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Fig. 3.   Substrate binding in P450BSbeta (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-sigma 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 (Neta 2 and Nepsilon atom) of Arg242. The distances between iron atom and atoms in palmitic acid is 4.9 Å (Fe-O), 5.1 Å (Fe-Calpha ), or 6.4 Å (Fe-Cbeta ).

As stated above, the substrate fatty acid could be stabilized in P450BSbeta 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 P450BSbeta 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 P450BSbeta . As a result of these specific interactions, the alpha - and beta -carbons are positioned near the iron sixth coordination site; 5.0 Å for Fe-Calpha and 6.2 Å for Fe-Cbeta . The configuration of the fatty acids (substrate) in P450BSbeta is entirely different from that in P450BM3 (11, 12) in which the omega -1, omega -2 and omega -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 P450BSbeta are discussed in the next section.

Site Specificity in Hydroxylation by P450 Peroxygenases-- As stated in the Introduction, P450SPalpha isolated from S. paucimobilis is, like P450BSbeta , 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 alpha -OH fatty acid is produced in the case of P450SPalpha , whereas 60% of the beta -OH and 40% of the alpha -OH fatty acids are produced in the case of P450BSbeta . 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 P450BSbeta corresponds to Arg241 in P450SPalpha , 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 P450BSbeta are substituted for Leu78 and Phe169 in P450SPalpha , respectively. To address their importance in determining the hydroxylation site specificity in peroxygenase P450s, we prepared three mutants of P450BSbeta in which Phe79 was replaced with leucine (F79L), Val170 with phenylalanine (V170F), and the double mutant (F79L/V170F).

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 P450BSbeta 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 (delta propionate), because this mode gives a band at 377 cm-1 for myoglobin (39). Indeed, the channel I for substrates in P450BSbeta 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 (nu 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 P450BSbeta . Indeed, in the Raman spectra of the R242A mutant, the position of both bands (delta propionate and nu Fe-CO) remained unchanged when substrate was added (data not shown).


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Fig. 4.   Resonance Raman spectra of P450BSbeta 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).

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 delta 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 nu 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 P450BSbeta , although the specific activity was reduced by half, the alpha /beta ratio changes from 1.4 to 0.2. In the reaction catalyzed by this mutant, the alpha -OH product is dominant.

                              
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Table II
Analyses of product and specific activities of P450BSbeta and its mutants (F79L, V171F, and F79L/V170F)

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 alpha /beta ratio was unaltered in the case of the V170F mutation, showing that these mutations are not suitable for the peroxygenase activity of P450BSbeta . In the resonance Raman spectra, the positions of the delta propionate bands of both mutants observed at 375 cm-1 hardly changes when the substrate fatty acid is added, although the position of the nu 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.

The mutation study suggested that Val170 directly affects the binding of a fatty acid to P450BSbeta 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 P450BSbeta is one of the important residues that control the alpha - or beta -site specificity of the hydroxylation reaction catalyzed by peroxygenases P450. In the crystal structure of P450BSbeta , 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.

Heme Active Site Structure and Possible Reaction Mechanism of Peroxygenase P450-- In the heme proximal side of P450BSbeta , 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 P450BSbeta , 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 P450BSbeta 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 P450BSbeta (stereo view).

The heme pocket structure of P450BSbeta 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 P450BSbeta 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 P450BSbeta , making for a quite polar heme pocket compared with the apolar heme pocket of P450BM3. This polar heme environment of P450BSbeta 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 P450BSbeta (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.

One of the most interesting findings in the active site structure of P450BSbeta 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 P450BSbeta that can connect the active site to the solvent region. Therefore, the heme active site of P450BSbeta 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 P450BSbeta , which does not require a proton supply during its catalytic reaction.

It should be noted here that the configuration of (Arg242-fatty acid carboxylate-H2O-Fe) observed in P450BSbeta 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 P450BSbeta as follows (see Scheme 1). The substrate-free enzyme of P450BSbeta (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 P450BSbeta 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 alpha - or beta -position, possibly according to a radical rebound mechanism, giving either the alpha - or beta -OH fatty acid as a product. In the crystal structure of the ferric enzyme, the alpha - and beta -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).


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Scheme 1.   Proposed mechanism of the P450BSbeta peroxygenase reaction.

One of the most striking and characteristic differences in the mechanism of P450BSbeta , 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 P450BSbeta , 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 epsilon -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.

Lastly, we discuss briefly the role of channel II of P450BSbeta , 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.

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 P450BSbeta can be clearly discussed on the basis of structural diversity that has been crystallographically determined.

    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.

    ABBREVIATIONS

The abbreviations used are: P450SPalpha , cytochrome P450 isolated from Sphingomonas paucimobilis; P450BSbeta , cytochrome P450 isolated from Bacillus subtilis; WT, wild type; MES, 4-morpholineethanesulfonic acid; CPO, chloroperoxidase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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