J Biol Inorg Chem DOI 10.1007/s00775-014-1106-9
MINIREVIEW
Peroxygenase reactions catalyzed by cytochromes P450 Osami Shoji • Yoshihito Watanabe
Received: 1 October 2013 / Accepted: 7 January 2014 Ó SBIC 2014
Abstract Cytochromes P450 (P450s) catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. By contrast with the general reductive oxygen activation pathway of P450s, an H2O2shunt pathway does not require any supply of electrons and protons for the generation of a highly reactive intermediate (compound I). Because the low cost of H2O2 allows us to use it in industrial-scale synthesis, the H2O2-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using H2O2 as the oxidant. Keywords Cytochrome Heme X-ray crystallography Site-directed mutagenesis Dioxygen Introduction Cytochromes P450 (P450s) are a family of heme–thiolate enzymes that catalyze monooxygenation of drugs,
Responsible editors: Lucia Banci and Claudio Luchinat. O. Shoji and Y. Watanabe equally contributed to this work. O. Shoji (&) Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan e-mail: [email protected]
detoxification of xenobiotics, and biosynthesis of steroids [1, 2]. One of the most interesting features of P450s is that they catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. Construction of biocatalysts based on P450s has therefore attracted much attention [1–11]. In general, P450s reductively activate molecular oxygen by using two electrons derived from a cofactor, NADH or NADPH, and two protons from outside the protein to generate an active species, ‘‘iron–oxo porphyrin p-cation radical,’’ which is called ‘‘compound I’’ after its discovery in peroxidases and catalases (Fig. 1) [12]. Hydrogen peroxide can also be used as an oxidant for the generation of the active species, through a ‘‘shunt reaction.’’ The use of H2O2 is an attractive option for monooxygenation reactions catalyzed by P450s, because electron transfer partners such as P450 reductase are not required for the generation of an active species. Furthermore, the low cost of H2O2 allows us to perform monooxygenations through the shunt path on an industrial scale. Thus, the H2O2-dependent P450 system has been considered as an important candidate for practical applications. This review focuses on the P450-catalyzed monooxygenation of organic molecules by using H2O2 as the oxidant. In the third section, artificial H2O2-dependent P450s created by point mutagenesis and directed evolution are described. In the final section, we summarize natural H2O2-dependent P450s and decoy molecule systems for peroxygenations of nonnative substrates.
H2O2-shunt reaction of P450s Y. Watanabe (&) Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan e-mail: [email protected]
P450s are monooxygenases, and their catalytic cycle of monooxygenation begins with a reductive activation of molecular oxygen by using two electrons derived from a
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J Biol Inorg Chem Fig. 1 a The crystal structure of P450 BM3 [Protein Data Bank (PDB) code 1FAG]. b The catalytic cycle of oxygen activation by cytochromes 450 (P450s) and its H2O2-shunt pathway
cofactor (NADH or NADPH) and two protons (Fig. 1). By contrast, P450s are known to precede an H2O2-shunt reaction where a supply of electrons and protons is not required for oxidation. In the H2O2-shunt reaction, the ferric heme iron reacts directly with H2O2 followed by a heterolytic cleavage of the O–O bond to give a highly reactive intermediate, so-called compound I that is the species responsible for the monooxygenation. The H2O2shunt reaction has been applied to a variety of oxidations that are commonly catalyzed by the usual P450 reaction system (Fig. 2). P450s examined include rabbit liver microsomal P450 [13, 14], rat liver microsomal P450 [15– 17], CYP119 [18], EpoK [19], CYP1A2 [20], CYP2S1 [21], CYP107AJ1 [22], CYP175A1 [23], P450eryF [24], P450st [25], CYP3A4 [26], and P450cam [27]. However, in general, the H2O2-shunt reaction is inefficient, and the catalytic activities of P450s are lower than those of reactions using cumene hydroperoxide or m-chloroperbenzoic acid [13, 28]. The crystal structures of P450s reported so far have revealed that amino acid residues that serve as general acid–base catalysts in peroxidases and catalase (histidine, aspartic acid, and glutamic acid) are not observed in the distal side of the heme (Fig. 3). General acid–base residues are crucial for the generation of compound I, which is responsible for H2O2-dependent oxidation (peroxidation). Indeed, most H2O2-dependent heme enzymes, such as horseradish peroxidase [29], chloroperoxidase [30, 31], aromatic peroxygenase [32–34], cytochrome c peroxidase [35], catalase-peroxidase [36], and catalase [37], have general acid–base residues close to the heme iron (Figs. 4, 5). The critical role of the general acid– base residues has been studied using a myoglobin
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framework. When a general acid–base residue is introduced into an appropriate position of the distal side of myoglobin by point mutagenesis, the formation of myoglobin compound I is accelerated, and higher peroxidase and peroxygenase activities are observed [38–40]. The lack of any general acid–base residue in the active site of P450s and the high hydrophobicity of the heme pocket of P450s result in low efficiency for the formation of compound I and the subsequent peroxidation. The distal side of most P450s is composed of hydrophobic amino acids residues, except for the conserved threonine. The conserved threonine is crucial for the reductive activation of molecular oxygen, and thus mutations of the conserved threonine in P450cam to alanine or valine resulted in H2O2 production (uncoupling of P450) rather than oxidation of the substrate [41, 42]. Although the conserved hydroxyl group in the active site of P450 is crucial for activating molecular oxygen as a proton donor, this alcohol side chain is not expected to serve as an acid–base catalyst for the facile generation of compound I when H2O2 is using as an oxidant, because the pKa of the alcohol side chain is too high.
Artificial H2O2-dependent P450s To construct artificial H2O2-dependent P450s, a variety of mutants (Figs. 6, 7) have been prepared by site-directed mutagenesis and random mutagenesis (directed evolution). Pioneering work in the development of artificial H2O2dependent P450s was reported by Li et al. [43] in 2001. They prepared a series of Phe-87 mutants of P450 BM3 (heme domain) and succeeded in oxidizing myristic acid
J Biol Inorg Chem Fig. 2 Peroxygenase reactions catalyzed by P450s: a benzphetamine [13], b aniline [14], c androstenedione [15], d methoxyresorufin [19], e 2amino-3-methylimidazo[4,5f]quinolone [20], f styrene [18], g 7,12dimethylbenz[a]anthracene [21], h palmitoleic acid [23], and i 7-ethoxycoumarin [22]. CYP cytochrome P450, WT wild type
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J Biol Inorg Chem Fig. 3 The active-site structures of a P450cam (PDB code 2CPP) [89], b CYP119 (PDB code 1F4T) [90], c CYP3A4 (PDB code 1TQN) [91], and d CYP175A1 (PDB code 1N97) [92]
Fig. 4 The active-site structures of horseradish peroxidase (PDB code1ATJ) [29], chloroperoxidase (PDB code 1CPO) [30, 31], aromatic peroxygenase (PDB code 2YP1) [32], cytochrome c peroxidase (PDB code 1CCA) [35], catalase-peroxidase (PDB code 1ITK) [36], and catalase (PDB code 4BLC) [37]
Fig. 5 General acid–base function of distal histidine in the formation of compound I using H2O2 as an oxidant
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and p-nitrophenoxydodecanoic acid [44] using H2O2 as an oxidant. Among the mutants examined, an F87A mutant showed the highest catalytic activity, 162 nmol/min/nmol P450. However, its Michaelis–Menten constant (Km) for H2O2 was estimated to be 24 mM, which is very high compared with those of P450s that naturally use H2O2 as an oxidant; for example, 50–72 lM for P450SPa [45] and 21 lM for P450BSb, respectively [46]. They also demonstrated the transformation of indole to indigo using P450 BM3 mutants and H2O2 [47], in which the F87V mutant afforded the highest activity. Cirino and Arnold [48] also used the F87V mutant of P450 BM3 and developed further improved mutants by applying a directed evolution technique (random mutation) based on the F87A mutant. In the peroxygenation of p-nitrophenoxydodecanoic acid, the initial turnover rate of the resulting mutant containing a nine amino acid substitution in addition to F87A (I58V, H100R, F107L, A135S, M145V, N239H, S274T, K434E, and V446I), named 21B3, was more than 18 times higher than that of the F87A mutant [49]. Its Michaelis–Menten constant for H2O2 was also improved from 24 mM to 10 mM. The 21B3 mutant also catalyzed styrene epoxidation and transformation of several pesticides such as linuron (Fig. 6e) [50]. Furthermore, the thermostability of 21B3 was improved by random mutagenesis. The resulting mutant, named 5H6 (L52I, I58V, F87A, H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and V446I), resists thermal denaturation and retains peroxygenase activity [51]. The hydroxylation activity of P450 BM3 for p-nitrophenoxydodecanoic acid [44] was improved by preparing chimeras of CYP102A1 (P450 BM3) with F87A substitution and CYP102A2 with F88A substitution [52, 53]. The turnover rate of a chimera named 169–197 (the first and last residues of CYP102A2 were inserted into CYP102A1) reached 100.3 nmol/min/ nmol P450 [52]. To prepare authentic human metabolites of propranolol [54], the P450 BM3 mutant 9C1 with 13 amino acid substitutions (F87A, I58V, H100R, I102T, F107L, A135S, M145A, N239H, S274T, L324I, I366V, K434E, E442K, and V446I) was constructed, and further mutations of seven active-site residues (A74, L75, V78, F81, A82, F87, T88) were conducted on the basis of the crystal structure of P450 BM3 containing N-palmitoylglycine in its active site [55]. The resulting mutant, named 2C11 (A74V, A82L, F87G, I58V, H100R, I102T, F107L, A135S, M145A, N239H, S274T, L324I, I366V, K434E, E442K, and V446I) showed enhanced activity for the formation of the authentic human metabolites of propranolol. It is interesting to note that a double mutant of 21B3 (W96A/F405L) gave a stable variant showing a higher peroxidase activity than the 21B3 mutant [56]. Joo et al. [57] developed an H2O2-dependent P450 BM3 and H2O2-
dependent P450cam using directed evolution. A triple mutant of P450cam (E331K, R280L, and C242F) expressed in Escherichia coli showed enhanced naphthalene hydroxylation activity [57], whereas the purified mutant showed not much improved catalytic activity [58]. Kumar et al. [59] applied a directed evolution approach to screen P450 2B1 for an H2O2-supported reaction and developed a mutant (V183L, F202L, L209A, and S334P) with a more than 30-fold higher kcat (7.2 min-1) than the wild type (0.2 min-1) for O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin. Many other mutants of P450 2B1 have been prepared, but kcat and Km (H2O2) of the resulting mutants such as L216W/F228I/T433S for the H2O2-supported reaction were not much improved [60]. Although many attempts have been devoted to improving the catalytic activities of P450 in the H2O2-shunt reaction by using mutants, there are not many mutants that show activities higher than those of the reductive oxygen activation process. It is noteworthy that any mutation introducing general acid–base residue(s) is not involved in artificial H2O2-dependent P450s. Mutations were sometimes introduced in places other than the active site. Therefore, no general acid–base residue in the active site was required for the facile generation of compound I in any artificial H2O2dependent P450s. These results suggest that water molecules exist in the active site and may serve as a general acid–base catalyst, even though further study is required to confirm the involvement of water molecules in the formation of compound I. If we could appropriately place an amino acid residue or residues that serve as a general acid–base catalyst in a suitable position in the active site of P450, we would obtain an artificial H2O2-dependent P450 with activity comparable to that of the natural P450s. It is interesting to note here that F87H, F87E, and F87D mutants of P450 BM3 were prepared to examine their effect on substrate selectivity and regioselectivity, but that the F87E and F87D mutants did not show any catalytic activity, whereas the F87H mutant showed a lower catalytic activity compared with the wild type. The CO difference spectra of the F87E and F87D mutants showed a peak at 420 nm indicating that these mutants are inactive forms; thus, they are unable to catalyze the monooxygenation through the general reductive oxygen activation pathway of P450s. We, however, presume that Phe-87 mutants would generate the active species through the H2O2-shunt pathway, because the location of Glu-87 on the distal side of P450 BM3 seems to be close enough to the heme iron to serve as a general acid–base catalyst. Similarly, we also presume that an A246E mutant of P450 BM3 would generate compound I by using H2O2, because the crystal structure of the heme domain of substrate-free A246E showed that the distance between an oxygen atom of the carboxylate group of Glu-246 and the heme iron is
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J Biol Inorg Chem Fig. 6 Peroxygenase reactions catalyzed by P450 BM3 mutants: a pnitrophenoxydodecanoic acid [43, 48, 51, 52], b indole [47], c styrene [49], d propranolol [54], and e linuron [50]
˚ , whereas another carboxylate group of Glu-246 in 5.6 A another molecule of the asymmetric unit is coordinated with the heme iron [61, 62].
Natural H2O2-dependent P450s and the decoy molecule system for nonnative substrate peroxygenations Although most P450s use the reductive molecular oxygen activation process for their monooxygenations, P450SPa (CYP152B1) [45, 63–67] from Sphingomonas paucimobilis, P450BSb (CYP152A1) [46, 68–73] from Bacillus subtilis, and P450CLA (CYP152A2) [74] from Clostridium acetobutylicum use H2O2 as the oxidant and catalyze
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hydroxylation of long-alkyl-chain fatty acids with high catalytic activities and high substrate specificity (Fig. 8). In addition, P450CLA catalyzes fatty acid hydroxylation through the reductive molecular oxygen activation pathway. Very recently, P450CLA and P450BSb were reported to perform peroxygenase reactions by the use of a light-driven system for in situ generation of H2O2 [75–78]. Among H2O2-dependent P450s, P450SPa reported by Matsunaga et al. [79] is the first P450 to be classified as an H2O2dependent P450. P450SPa catalyzes a-selective (100 %) hydroxylation of long-alkyl-chain fatty acids such as myristic acid and palmitic acid. In 2003, the crystal structure of P450BSb (CYP152A1) was reported at a reso˚ (Protein Data Bank code 1IZO) as the first lution of 2.1 A
J Biol Inorg Chem Fig. 7 Peroxygenase reactions catalyzed by P450 mutants: a naphthalene [57], b 7-ethoxy4-trifluoromethylcoumarin [59], and c 7-benzyloxyquinoline [60]
Fig. 8 Myristic acid hydroxylation catalyzed by H2O2-dependent P450s: a P450BSb [80], b P450SPa [80], and c P450CLA [74]
crystal structure of an H2O2-dependent P450 [69]. P450BSb has 42 % amino acid identity with P450SPa and oxidizes the a and b positions of fatty acids in a roughly 40:60 ratio. The crystal structure of a substrate-bound form of P450BSb revealed that P450BSb lacks general acid–base residues around the distal side of the heme, although general acid– base residues are highly conserved in peroxidases and peroxygenases, as described in ‘‘H2O2-shunt reaction of P450s.’’ Instead of the general acid–base residues, the terminal carboxylate group of the bound fatty acid interacts with the guanidinium group of Arg-242 located near the heme group (Fig. 9b). The distance between an oxygen atom of the carboxylate group of palmitic acid and the
˚ , which is very close to that of aromatic heme iron is 5.3 A ˚ ) [32] and chloroperoxidase (5.1 A ˚) peroxygenase (5.2 A [30, 31] (Fig. 4). Furthermore, the resulting salt bridge in the active site is very similar to that of aromatic peroxygenase [32]. The location of the carboxylate group of palmitic acid bound to P450BSb suggests that a general acid–base function for the facile formation of compound I is accomplished by the carboxylate group of the substrate (Fig. 9). Thus, the catalytic reaction begins with the fixation of a substrate by the interaction of the terminal carboxyl group of the fatty acid with Arg-242. The crystal structure of P450SPa con˚ (Protein Data taining palmitic acid at a resolution of 1.65 A Bank code 3AWM) was determined in 2011 [80]. The structure also revealed that the carboxylate group of the fatty acid interacts with Arg-241 located immediately above the heme. Two crystal structural studies confirm that the substrate-assisted activation mechanism is conserved in H2O2-dependent P450s. This unique reaction mechanism observed for P450BSb and P450SPa also contributes to their high substrate specificity. P450BSb and P450SPa do not oxidize substrates other than long-alkyl-chain fatty acids. For example, P450BSb never oxidizes substrates without a carboxyl group such as tetradecane, 1-tetradecanol, and tetradecanal. The fatty acid substrate itself serves both as an initiator and as an activator of the reaction, and thus P450SPa and P450BSb never oxidize substrates other than fatty acids.
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Fig. 9 a A proposed catalytic hydroxylation mechanism for P450BSb and the roles of the substrate carboxylate–Arg-242 salt bridge. b The active-site structure of P450BSb containing palmitic acid (PDB code 1IZO). c The substrate misrecognition system in the oxidation of nonnative substrate (e.g., ethylbenzene) in the presence of heptanoic
acid as a decoy molecule. d The active-site structure of P450BSb cocrystallized with heptanoic acid (PDB code 2ZQX). An acetic acid molecule was placed in the electron density observed near the Arg242 residue
Recently, we found that P450BSb is able to oxidize a wide variety of nonnative substrates in the presence of a series of short-alkyl-chain carboxylic acids (C4–C10), which we call ‘‘decoy molecules’’ [81]. The peroxygenation of nonnative substrates never proceeds without decoy molecules, and thus these reactions are catalyzed by P450BSb with the help of these short-alkyl-chain carboxylic acids (Fig. 9c). Namely, P450BSb misrecognizes decoy molecules as its native substrates (long-alkyl-chain fatty acids) and the resulting salt bridge (Arg-242–carboxylate of fatty acid) serves as a general acid–base catalyst to allow the formation of an active species followed by the oxidation of nonnative substrates. The role of decoy molecules was confirmed on the basis of crystal structure analysis of a heptanoic acid bound form of P450BSb (Fig. 9d) [82]. A clear electron density assignable to the carboxylate group of heptanoic acid was observed in the active site, suggesting the participation of the carboxylate group of decoy molecules in the formation of compound I. Later, a clear structure of decoy-molecule-bound P450SPa was obtained, when (R)-ibuprofen was used as the decoy molecule (Fig. 10) [83]. By use of a simple substrate misrecognition
trick by using decoy molecules, nonnative substrate oxidations such as the one-electron oxidation of guaiacol [81], sulfoxidation of thioanisole [84], epoxidation of styrene [81], C–H bond hydroxylation of ethylbenzene [81], and aromatic C–H bond hydroxylation of 1-methoxynaphthalene [85] were catalyzed by P450BSb (Tables 1, 2). Interestingly, the catalytic activities are very much dependent on the alkyl-chain length of decoy molecules (Table 1). Furthermore, enantioselectivity in peroxygenation such as ethylbenzene hydroxylation is dependent on the structure of the decoy molecules used [81]. These results clearly show that the decoy molecule plays two important roles: (1) activation of P450BSb to form the active species compound I, and (2) regulation of the spatial orientation of the substrate. Therefore, the decoy molecule system can be applicable to synthetic chemistry. In the case of P450SPa, the stereoselectivity of styrene epoxidation was also controlled by the chirality of the decoy molecules (Fig. 10) [83]; whereas (R)-ibuprofen gave (S)-styrene oxide, (S)ibuprofen gave (R)-styrene oxide. More importantly, the enantioselectivity was predicted by docking simulation based on the structure of P450SPa containing (R)-ibuprofen
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J Biol Inorg Chem Fig. 10 a Stereoselective epoxidation of styrene catalyzed by P450SPa in the presence of (R)-ibuprofen or S)-ibuprofen. b The active-site structure of the (R)-ibuprofen-bound form of P450SPa (PDB code 3VM4). ee% percent enantiomeric excess
Table 1 The effect of decoy molecules on the one-electron oxidation of guaiacol O
O OH
P450BSβ, H2O2
O
O O
Decoy molecules
O O
Decoy molecule
Turnover number/min
None
0
Acetic acid (2)
0
Propionic acid (3)
26
Butanoic acid (4)
230
Pentanoic acid (5)
1,900
Hexanoic acid (6)
2,420
Heptanoic acid (7)
3,750
Octanoic acid (8)
2,490
Nonanoic acid (9)
2,380
Decanoic acid (10)
2,360
Myristic acid (14)
14
Table 2 The reactions catalyzed by P450BSb in the presence of decoy molecules
The initial turnover rates in the presence and in the absence of carboxylic acids. The numbers in parentheses denote the number of carbon atoms.
[83]. The decoy molecule system does not require any substitution of amino acids to alter the substrate specificity of P450BSb or to alter the enantioselectivity of nonnative substrate oxidations. The decoy molecule system has also been applied to P450 BM3 in which a series of perfluorinated carboxylic acids serve as decoy molecules [86]. The ‘‘P450BM3-decoy molecule system’’ catalyzed gaseous alkane hydroxylation such as propane [86] and ethane [87] as well as benzene [88], whereas wild-type P450 BM3 never catalyzes these reactions.
Conclusions and future prospects In this review, we have described H2O2-dependent oxidations (peroxygenations) catalyzed by P450s. The H2O2shunt reaction of P450s, as practical biocatalysts, has been
considered as an attractive reaction system for the monooxygenation. The catalytic activities of most H2O2-shunt reactions, however, are lower than those of the natural reductive oxygen activation processes of P450s. The catalytic activities of H2O2-dependent reactions can be improved by mutagenesis. Poor activities of P450 mutants for H2O2-dependent reactions could be explained by the lack of general acid–base residues in the active site of P450s and their mutants. By contrast, for the natural H2O2dependent P450s, the general acid–base catalysts supplied by natural substrates (fatty acids) or decoy molecules greatly contribute to their high catalytic activities. These findings lead us to the conclusion that most P450s can be converted into efficient H2O2-dependent P450s, either by placing a general acid–base residue(s) at an appropriate position in the active site of P450s by mutagenesis or by
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adding external molecules, such as decoy molecules, as an activator, or both. Acknowledgments This work was supported, in part, by a Grant-inAid for Scientific Research (S) to Y. W. (24225004), a Grant-in-Aid for Young Scientists (A) to O. S. (21685018), and a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Molecular Activation Directed Toward Straightforward Synthesis’’ to O.S. (25105724) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan).
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MINIREVIEW
Peroxygenase reactions catalyzed by cytochromes P450 Osami Shoji • Yoshihito Watanabe
Received: 1 October 2013 / Accepted: 7 January 2014 Ó SBIC 2014
Abstract Cytochromes P450 (P450s) catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. By contrast with the general reductive oxygen activation pathway of P450s, an H2O2shunt pathway does not require any supply of electrons and protons for the generation of a highly reactive intermediate (compound I). Because the low cost of H2O2 allows us to use it in industrial-scale synthesis, the H2O2-shunt pathway is an attractive process for monooxygenation reactions. This review focuses on the P450-catalyzed monooxygenation of organic molecules using H2O2 as the oxidant. Keywords Cytochrome Heme X-ray crystallography Site-directed mutagenesis Dioxygen Introduction Cytochromes P450 (P450s) are a family of heme–thiolate enzymes that catalyze monooxygenation of drugs,
Responsible editors: Lucia Banci and Claudio Luchinat. O. Shoji and Y. Watanabe equally contributed to this work. O. Shoji (&) Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan e-mail: [email protected]
detoxification of xenobiotics, and biosynthesis of steroids [1, 2]. One of the most interesting features of P450s is that they catalyze monooxygenation of a wide range of less reactive organic molecules under mild conditions. Construction of biocatalysts based on P450s has therefore attracted much attention [1–11]. In general, P450s reductively activate molecular oxygen by using two electrons derived from a cofactor, NADH or NADPH, and two protons from outside the protein to generate an active species, ‘‘iron–oxo porphyrin p-cation radical,’’ which is called ‘‘compound I’’ after its discovery in peroxidases and catalases (Fig. 1) [12]. Hydrogen peroxide can also be used as an oxidant for the generation of the active species, through a ‘‘shunt reaction.’’ The use of H2O2 is an attractive option for monooxygenation reactions catalyzed by P450s, because electron transfer partners such as P450 reductase are not required for the generation of an active species. Furthermore, the low cost of H2O2 allows us to perform monooxygenations through the shunt path on an industrial scale. Thus, the H2O2-dependent P450 system has been considered as an important candidate for practical applications. This review focuses on the P450-catalyzed monooxygenation of organic molecules by using H2O2 as the oxidant. In the third section, artificial H2O2-dependent P450s created by point mutagenesis and directed evolution are described. In the final section, we summarize natural H2O2-dependent P450s and decoy molecule systems for peroxygenations of nonnative substrates.
H2O2-shunt reaction of P450s Y. Watanabe (&) Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan e-mail: [email protected]
P450s are monooxygenases, and their catalytic cycle of monooxygenation begins with a reductive activation of molecular oxygen by using two electrons derived from a
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J Biol Inorg Chem Fig. 1 a The crystal structure of P450 BM3 [Protein Data Bank (PDB) code 1FAG]. b The catalytic cycle of oxygen activation by cytochromes 450 (P450s) and its H2O2-shunt pathway
cofactor (NADH or NADPH) and two protons (Fig. 1). By contrast, P450s are known to precede an H2O2-shunt reaction where a supply of electrons and protons is not required for oxidation. In the H2O2-shunt reaction, the ferric heme iron reacts directly with H2O2 followed by a heterolytic cleavage of the O–O bond to give a highly reactive intermediate, so-called compound I that is the species responsible for the monooxygenation. The H2O2shunt reaction has been applied to a variety of oxidations that are commonly catalyzed by the usual P450 reaction system (Fig. 2). P450s examined include rabbit liver microsomal P450 [13, 14], rat liver microsomal P450 [15– 17], CYP119 [18], EpoK [19], CYP1A2 [20], CYP2S1 [21], CYP107AJ1 [22], CYP175A1 [23], P450eryF [24], P450st [25], CYP3A4 [26], and P450cam [27]. However, in general, the H2O2-shunt reaction is inefficient, and the catalytic activities of P450s are lower than those of reactions using cumene hydroperoxide or m-chloroperbenzoic acid [13, 28]. The crystal structures of P450s reported so far have revealed that amino acid residues that serve as general acid–base catalysts in peroxidases and catalase (histidine, aspartic acid, and glutamic acid) are not observed in the distal side of the heme (Fig. 3). General acid–base residues are crucial for the generation of compound I, which is responsible for H2O2-dependent oxidation (peroxidation). Indeed, most H2O2-dependent heme enzymes, such as horseradish peroxidase [29], chloroperoxidase [30, 31], aromatic peroxygenase [32–34], cytochrome c peroxidase [35], catalase-peroxidase [36], and catalase [37], have general acid–base residues close to the heme iron (Figs. 4, 5). The critical role of the general acid– base residues has been studied using a myoglobin
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framework. When a general acid–base residue is introduced into an appropriate position of the distal side of myoglobin by point mutagenesis, the formation of myoglobin compound I is accelerated, and higher peroxidase and peroxygenase activities are observed [38–40]. The lack of any general acid–base residue in the active site of P450s and the high hydrophobicity of the heme pocket of P450s result in low efficiency for the formation of compound I and the subsequent peroxidation. The distal side of most P450s is composed of hydrophobic amino acids residues, except for the conserved threonine. The conserved threonine is crucial for the reductive activation of molecular oxygen, and thus mutations of the conserved threonine in P450cam to alanine or valine resulted in H2O2 production (uncoupling of P450) rather than oxidation of the substrate [41, 42]. Although the conserved hydroxyl group in the active site of P450 is crucial for activating molecular oxygen as a proton donor, this alcohol side chain is not expected to serve as an acid–base catalyst for the facile generation of compound I when H2O2 is using as an oxidant, because the pKa of the alcohol side chain is too high.
Artificial H2O2-dependent P450s To construct artificial H2O2-dependent P450s, a variety of mutants (Figs. 6, 7) have been prepared by site-directed mutagenesis and random mutagenesis (directed evolution). Pioneering work in the development of artificial H2O2dependent P450s was reported by Li et al. [43] in 2001. They prepared a series of Phe-87 mutants of P450 BM3 (heme domain) and succeeded in oxidizing myristic acid
J Biol Inorg Chem Fig. 2 Peroxygenase reactions catalyzed by P450s: a benzphetamine [13], b aniline [14], c androstenedione [15], d methoxyresorufin [19], e 2amino-3-methylimidazo[4,5f]quinolone [20], f styrene [18], g 7,12dimethylbenz[a]anthracene [21], h palmitoleic acid [23], and i 7-ethoxycoumarin [22]. CYP cytochrome P450, WT wild type
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J Biol Inorg Chem Fig. 3 The active-site structures of a P450cam (PDB code 2CPP) [89], b CYP119 (PDB code 1F4T) [90], c CYP3A4 (PDB code 1TQN) [91], and d CYP175A1 (PDB code 1N97) [92]
Fig. 4 The active-site structures of horseradish peroxidase (PDB code1ATJ) [29], chloroperoxidase (PDB code 1CPO) [30, 31], aromatic peroxygenase (PDB code 2YP1) [32], cytochrome c peroxidase (PDB code 1CCA) [35], catalase-peroxidase (PDB code 1ITK) [36], and catalase (PDB code 4BLC) [37]
Fig. 5 General acid–base function of distal histidine in the formation of compound I using H2O2 as an oxidant
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and p-nitrophenoxydodecanoic acid [44] using H2O2 as an oxidant. Among the mutants examined, an F87A mutant showed the highest catalytic activity, 162 nmol/min/nmol P450. However, its Michaelis–Menten constant (Km) for H2O2 was estimated to be 24 mM, which is very high compared with those of P450s that naturally use H2O2 as an oxidant; for example, 50–72 lM for P450SPa [45] and 21 lM for P450BSb, respectively [46]. They also demonstrated the transformation of indole to indigo using P450 BM3 mutants and H2O2 [47], in which the F87V mutant afforded the highest activity. Cirino and Arnold [48] also used the F87V mutant of P450 BM3 and developed further improved mutants by applying a directed evolution technique (random mutation) based on the F87A mutant. In the peroxygenation of p-nitrophenoxydodecanoic acid, the initial turnover rate of the resulting mutant containing a nine amino acid substitution in addition to F87A (I58V, H100R, F107L, A135S, M145V, N239H, S274T, K434E, and V446I), named 21B3, was more than 18 times higher than that of the F87A mutant [49]. Its Michaelis–Menten constant for H2O2 was also improved from 24 mM to 10 mM. The 21B3 mutant also catalyzed styrene epoxidation and transformation of several pesticides such as linuron (Fig. 6e) [50]. Furthermore, the thermostability of 21B3 was improved by random mutagenesis. The resulting mutant, named 5H6 (L52I, I58V, F87A, H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and V446I), resists thermal denaturation and retains peroxygenase activity [51]. The hydroxylation activity of P450 BM3 for p-nitrophenoxydodecanoic acid [44] was improved by preparing chimeras of CYP102A1 (P450 BM3) with F87A substitution and CYP102A2 with F88A substitution [52, 53]. The turnover rate of a chimera named 169–197 (the first and last residues of CYP102A2 were inserted into CYP102A1) reached 100.3 nmol/min/ nmol P450 [52]. To prepare authentic human metabolites of propranolol [54], the P450 BM3 mutant 9C1 with 13 amino acid substitutions (F87A, I58V, H100R, I102T, F107L, A135S, M145A, N239H, S274T, L324I, I366V, K434E, E442K, and V446I) was constructed, and further mutations of seven active-site residues (A74, L75, V78, F81, A82, F87, T88) were conducted on the basis of the crystal structure of P450 BM3 containing N-palmitoylglycine in its active site [55]. The resulting mutant, named 2C11 (A74V, A82L, F87G, I58V, H100R, I102T, F107L, A135S, M145A, N239H, S274T, L324I, I366V, K434E, E442K, and V446I) showed enhanced activity for the formation of the authentic human metabolites of propranolol. It is interesting to note that a double mutant of 21B3 (W96A/F405L) gave a stable variant showing a higher peroxidase activity than the 21B3 mutant [56]. Joo et al. [57] developed an H2O2-dependent P450 BM3 and H2O2-
dependent P450cam using directed evolution. A triple mutant of P450cam (E331K, R280L, and C242F) expressed in Escherichia coli showed enhanced naphthalene hydroxylation activity [57], whereas the purified mutant showed not much improved catalytic activity [58]. Kumar et al. [59] applied a directed evolution approach to screen P450 2B1 for an H2O2-supported reaction and developed a mutant (V183L, F202L, L209A, and S334P) with a more than 30-fold higher kcat (7.2 min-1) than the wild type (0.2 min-1) for O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin. Many other mutants of P450 2B1 have been prepared, but kcat and Km (H2O2) of the resulting mutants such as L216W/F228I/T433S for the H2O2-supported reaction were not much improved [60]. Although many attempts have been devoted to improving the catalytic activities of P450 in the H2O2-shunt reaction by using mutants, there are not many mutants that show activities higher than those of the reductive oxygen activation process. It is noteworthy that any mutation introducing general acid–base residue(s) is not involved in artificial H2O2-dependent P450s. Mutations were sometimes introduced in places other than the active site. Therefore, no general acid–base residue in the active site was required for the facile generation of compound I in any artificial H2O2dependent P450s. These results suggest that water molecules exist in the active site and may serve as a general acid–base catalyst, even though further study is required to confirm the involvement of water molecules in the formation of compound I. If we could appropriately place an amino acid residue or residues that serve as a general acid–base catalyst in a suitable position in the active site of P450, we would obtain an artificial H2O2-dependent P450 with activity comparable to that of the natural P450s. It is interesting to note here that F87H, F87E, and F87D mutants of P450 BM3 were prepared to examine their effect on substrate selectivity and regioselectivity, but that the F87E and F87D mutants did not show any catalytic activity, whereas the F87H mutant showed a lower catalytic activity compared with the wild type. The CO difference spectra of the F87E and F87D mutants showed a peak at 420 nm indicating that these mutants are inactive forms; thus, they are unable to catalyze the monooxygenation through the general reductive oxygen activation pathway of P450s. We, however, presume that Phe-87 mutants would generate the active species through the H2O2-shunt pathway, because the location of Glu-87 on the distal side of P450 BM3 seems to be close enough to the heme iron to serve as a general acid–base catalyst. Similarly, we also presume that an A246E mutant of P450 BM3 would generate compound I by using H2O2, because the crystal structure of the heme domain of substrate-free A246E showed that the distance between an oxygen atom of the carboxylate group of Glu-246 and the heme iron is
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J Biol Inorg Chem Fig. 6 Peroxygenase reactions catalyzed by P450 BM3 mutants: a pnitrophenoxydodecanoic acid [43, 48, 51, 52], b indole [47], c styrene [49], d propranolol [54], and e linuron [50]
˚ , whereas another carboxylate group of Glu-246 in 5.6 A another molecule of the asymmetric unit is coordinated with the heme iron [61, 62].
Natural H2O2-dependent P450s and the decoy molecule system for nonnative substrate peroxygenations Although most P450s use the reductive molecular oxygen activation process for their monooxygenations, P450SPa (CYP152B1) [45, 63–67] from Sphingomonas paucimobilis, P450BSb (CYP152A1) [46, 68–73] from Bacillus subtilis, and P450CLA (CYP152A2) [74] from Clostridium acetobutylicum use H2O2 as the oxidant and catalyze
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hydroxylation of long-alkyl-chain fatty acids with high catalytic activities and high substrate specificity (Fig. 8). In addition, P450CLA catalyzes fatty acid hydroxylation through the reductive molecular oxygen activation pathway. Very recently, P450CLA and P450BSb were reported to perform peroxygenase reactions by the use of a light-driven system for in situ generation of H2O2 [75–78]. Among H2O2-dependent P450s, P450SPa reported by Matsunaga et al. [79] is the first P450 to be classified as an H2O2dependent P450. P450SPa catalyzes a-selective (100 %) hydroxylation of long-alkyl-chain fatty acids such as myristic acid and palmitic acid. In 2003, the crystal structure of P450BSb (CYP152A1) was reported at a reso˚ (Protein Data Bank code 1IZO) as the first lution of 2.1 A
J Biol Inorg Chem Fig. 7 Peroxygenase reactions catalyzed by P450 mutants: a naphthalene [57], b 7-ethoxy4-trifluoromethylcoumarin [59], and c 7-benzyloxyquinoline [60]
Fig. 8 Myristic acid hydroxylation catalyzed by H2O2-dependent P450s: a P450BSb [80], b P450SPa [80], and c P450CLA [74]
crystal structure of an H2O2-dependent P450 [69]. P450BSb has 42 % amino acid identity with P450SPa and oxidizes the a and b positions of fatty acids in a roughly 40:60 ratio. The crystal structure of a substrate-bound form of P450BSb revealed that P450BSb lacks general acid–base residues around the distal side of the heme, although general acid– base residues are highly conserved in peroxidases and peroxygenases, as described in ‘‘H2O2-shunt reaction of P450s.’’ Instead of the general acid–base residues, the terminal carboxylate group of the bound fatty acid interacts with the guanidinium group of Arg-242 located near the heme group (Fig. 9b). The distance between an oxygen atom of the carboxylate group of palmitic acid and the
˚ , which is very close to that of aromatic heme iron is 5.3 A ˚ ) [32] and chloroperoxidase (5.1 A ˚) peroxygenase (5.2 A [30, 31] (Fig. 4). Furthermore, the resulting salt bridge in the active site is very similar to that of aromatic peroxygenase [32]. The location of the carboxylate group of palmitic acid bound to P450BSb suggests that a general acid–base function for the facile formation of compound I is accomplished by the carboxylate group of the substrate (Fig. 9). Thus, the catalytic reaction begins with the fixation of a substrate by the interaction of the terminal carboxyl group of the fatty acid with Arg-242. The crystal structure of P450SPa con˚ (Protein Data taining palmitic acid at a resolution of 1.65 A Bank code 3AWM) was determined in 2011 [80]. The structure also revealed that the carboxylate group of the fatty acid interacts with Arg-241 located immediately above the heme. Two crystal structural studies confirm that the substrate-assisted activation mechanism is conserved in H2O2-dependent P450s. This unique reaction mechanism observed for P450BSb and P450SPa also contributes to their high substrate specificity. P450BSb and P450SPa do not oxidize substrates other than long-alkyl-chain fatty acids. For example, P450BSb never oxidizes substrates without a carboxyl group such as tetradecane, 1-tetradecanol, and tetradecanal. The fatty acid substrate itself serves both as an initiator and as an activator of the reaction, and thus P450SPa and P450BSb never oxidize substrates other than fatty acids.
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Fig. 9 a A proposed catalytic hydroxylation mechanism for P450BSb and the roles of the substrate carboxylate–Arg-242 salt bridge. b The active-site structure of P450BSb containing palmitic acid (PDB code 1IZO). c The substrate misrecognition system in the oxidation of nonnative substrate (e.g., ethylbenzene) in the presence of heptanoic
acid as a decoy molecule. d The active-site structure of P450BSb cocrystallized with heptanoic acid (PDB code 2ZQX). An acetic acid molecule was placed in the electron density observed near the Arg242 residue
Recently, we found that P450BSb is able to oxidize a wide variety of nonnative substrates in the presence of a series of short-alkyl-chain carboxylic acids (C4–C10), which we call ‘‘decoy molecules’’ [81]. The peroxygenation of nonnative substrates never proceeds without decoy molecules, and thus these reactions are catalyzed by P450BSb with the help of these short-alkyl-chain carboxylic acids (Fig. 9c). Namely, P450BSb misrecognizes decoy molecules as its native substrates (long-alkyl-chain fatty acids) and the resulting salt bridge (Arg-242–carboxylate of fatty acid) serves as a general acid–base catalyst to allow the formation of an active species followed by the oxidation of nonnative substrates. The role of decoy molecules was confirmed on the basis of crystal structure analysis of a heptanoic acid bound form of P450BSb (Fig. 9d) [82]. A clear electron density assignable to the carboxylate group of heptanoic acid was observed in the active site, suggesting the participation of the carboxylate group of decoy molecules in the formation of compound I. Later, a clear structure of decoy-molecule-bound P450SPa was obtained, when (R)-ibuprofen was used as the decoy molecule (Fig. 10) [83]. By use of a simple substrate misrecognition
trick by using decoy molecules, nonnative substrate oxidations such as the one-electron oxidation of guaiacol [81], sulfoxidation of thioanisole [84], epoxidation of styrene [81], C–H bond hydroxylation of ethylbenzene [81], and aromatic C–H bond hydroxylation of 1-methoxynaphthalene [85] were catalyzed by P450BSb (Tables 1, 2). Interestingly, the catalytic activities are very much dependent on the alkyl-chain length of decoy molecules (Table 1). Furthermore, enantioselectivity in peroxygenation such as ethylbenzene hydroxylation is dependent on the structure of the decoy molecules used [81]. These results clearly show that the decoy molecule plays two important roles: (1) activation of P450BSb to form the active species compound I, and (2) regulation of the spatial orientation of the substrate. Therefore, the decoy molecule system can be applicable to synthetic chemistry. In the case of P450SPa, the stereoselectivity of styrene epoxidation was also controlled by the chirality of the decoy molecules (Fig. 10) [83]; whereas (R)-ibuprofen gave (S)-styrene oxide, (S)ibuprofen gave (R)-styrene oxide. More importantly, the enantioselectivity was predicted by docking simulation based on the structure of P450SPa containing (R)-ibuprofen
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J Biol Inorg Chem Fig. 10 a Stereoselective epoxidation of styrene catalyzed by P450SPa in the presence of (R)-ibuprofen or S)-ibuprofen. b The active-site structure of the (R)-ibuprofen-bound form of P450SPa (PDB code 3VM4). ee% percent enantiomeric excess
Table 1 The effect of decoy molecules on the one-electron oxidation of guaiacol O
O OH
P450BSβ, H2O2
O
O O
Decoy molecules
O O
Decoy molecule
Turnover number/min
None
0
Acetic acid (2)
0
Propionic acid (3)
26
Butanoic acid (4)
230
Pentanoic acid (5)
1,900
Hexanoic acid (6)
2,420
Heptanoic acid (7)
3,750
Octanoic acid (8)
2,490
Nonanoic acid (9)
2,380
Decanoic acid (10)
2,360
Myristic acid (14)
14
Table 2 The reactions catalyzed by P450BSb in the presence of decoy molecules
The initial turnover rates in the presence and in the absence of carboxylic acids. The numbers in parentheses denote the number of carbon atoms.
[83]. The decoy molecule system does not require any substitution of amino acids to alter the substrate specificity of P450BSb or to alter the enantioselectivity of nonnative substrate oxidations. The decoy molecule system has also been applied to P450 BM3 in which a series of perfluorinated carboxylic acids serve as decoy molecules [86]. The ‘‘P450BM3-decoy molecule system’’ catalyzed gaseous alkane hydroxylation such as propane [86] and ethane [87] as well as benzene [88], whereas wild-type P450 BM3 never catalyzes these reactions.
Conclusions and future prospects In this review, we have described H2O2-dependent oxidations (peroxygenations) catalyzed by P450s. The H2O2shunt reaction of P450s, as practical biocatalysts, has been
considered as an attractive reaction system for the monooxygenation. The catalytic activities of most H2O2-shunt reactions, however, are lower than those of the natural reductive oxygen activation processes of P450s. The catalytic activities of H2O2-dependent reactions can be improved by mutagenesis. Poor activities of P450 mutants for H2O2-dependent reactions could be explained by the lack of general acid–base residues in the active site of P450s and their mutants. By contrast, for the natural H2O2dependent P450s, the general acid–base catalysts supplied by natural substrates (fatty acids) or decoy molecules greatly contribute to their high catalytic activities. These findings lead us to the conclusion that most P450s can be converted into efficient H2O2-dependent P450s, either by placing a general acid–base residue(s) at an appropriate position in the active site of P450s by mutagenesis or by
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adding external molecules, such as decoy molecules, as an activator, or both. Acknowledgments This work was supported, in part, by a Grant-inAid for Scientific Research (S) to Y. W. (24225004), a Grant-in-Aid for Young Scientists (A) to O. S. (21685018), and a Grant-in-Aid for Scientific Research on Innovative Areas ‘‘Molecular Activation Directed Toward Straightforward Synthesis’’ to O.S. (25105724) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan).
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