Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native

Article Cite This: Acc. Chem. Res. XXXX, XXX, XXX−XXX

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Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native Substrates by Exploiting Its Substrate Misrecognition Published as part of the Accounts of Chemical Research special issue “Artificial Metalloenzymes and Abiological Catalysis of Metalloenzymes”.

Osami Shoji* Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Acc. Chem. Res. Downloaded from pubs.acs.org by UNIV OF READING on 03/19/19. For personal use only.

Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, 5 Sanbancho, Chiyoda-ku, Tokyo 102-0075, Japan

Yuichiro Aiba Department of Chemistry, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

Yoshihito Watanabe* Research Center for Materials Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan

CONSPECTUS: Bacterial cytochrome P450s (P450s) are at the focus of attention as potential biocatalysts for applications in green synthetic chemistry, as they possess high activity for the hydroxylation of inert substrate C−H bonds. The high activity of bacterial P450s, such as P450BM3, is chiefly due to their high substrate specificity, and consequently, the catalytic activity of P450BM3 toward non-native substrates is very low, limiting the utility of bacterial P450s as biocatalysts. To enable oxidation of non-native substrates by P450BM3 without any mutagenesis, we have developed a series of “decoy molecules”, inert dummy substrates, with structures that resemble those of the native substrates. Decoy molecules fool P450BM3 into generating the active species, so-called Compound I, enabling the catalytic oxidation of non-native substrates other than fatty acids. Perfluorinated carboxylic acids (PFCs) serve as decoy molecules to initiate the activation of molecular oxygen in the same manner as long-alkyl-chain fatty acids, due to their structural similarity, and induce the generation of Compound I, but, unlike the native substrates, PFCs are not oxidizable by Compound I, allowing the hydroxylation of non-native substrates, such as gaseous alkanes and benzene. The catalytic activity for non-native substrate hydroxylation was significantly enhanced by employing second generation decoy molecules, PFCs modified with amino acids (PFC-amino acids). Cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty-acid-binding channel that was readily assigned to PFC9-Trp. The alkyl chain terminus of PFC9-Trp does not reach the active site owing to multiple hydrogen bonding interactions between the carboxyl and carbonyl groups of PFC9-Trp and amino acids located at the entrance of the substrate binding channel of P450BM3 that fix it in place. The remaining space above the heme after binding of PFC9-Trp can be utilized to accommodate non-native substrates. Further developments revealed that third generation decoy molecules, N-acyl amino acids, such as pelargonoyl-L-phenylalanine (C9-Phe), can serve as decoy molecules, indicating that the rationale “fluorination is required for decoy molecule function” can be safely discarded. Diverse carboxylic acids including dipeptides could now be exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the third-generation decoy molecules examined N-enanthyl-L-proline modified with L-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation. The structural diversity of thirdgeneration decoy molecules was also utilized to control the stereoselectivity of hydroxylation for the benzylic hydroxylation of Indane, showing that decoy molecules can alter stereoselectivity. As both the catalytic activity and enantioselectivity are dependent upon the structure of the decoy molecules, their design allows us to regulate reactions catalyzed by wild-type enzymes. Furthermore, decoy molecules can also activate intracellular P450BM3, allowing the use of E. coli expressing wild-type P450BM3 as an efficient whole-cell bioreactor. It should be noted that Mn-substituted full-length P450BM3 (Mn-P450BM3) is also active for the hydroxylation of propane in which the regioselectivity diverged from that of FeP450BM3. The results summarized in this Account represent good examples of how the reactive properties of P450BM3 can be controlled for the monooxygenation of non-native substrates in vitro as well as in vivo to expand the potential of P450BM3.

Received: December 20, 2018

© XXXX American Chemical Society

A

DOI: 10.1021/acs.accounts.8b00651 Acc. Chem. Res. XXXX, XXX, XXX−XXX

Article

Accounts of Chemical Research

Figure 1. Catalytic cycle of cytochrome P450.

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successful at catalyzing the hydroxylation of gaseous alkanes, such as ethane and propane,13−17 demonstrating that the mutagenesis of P450s to construct a binding pocket suitable for non-native substrates is a promising approach. However, we proposed a simple and unique strategy to hydroxylate nonnative substrates by using wild-type P450BM3. Instead of mutagenesis, we utilized inert dummy substrates (“decoy molecules”), structural analogs of the natural substrates. We envisioned that the binding of these decoy molecules should be sufficient to remodel the size and shape of P450BM3’s active site, enabling even wild-type P450BM3 to accommodate nonnative substrates and oxidize them, thereby removing the need for laborious mutagenesis. This review focuses on the development of decoy molecules and how they can be used to activate wild-type P450BM3 for the monooxygenation of non-native substrates in vitro and in vivo.

INTRODUCTION Cytochrome P450s (P450s or CYPs) represent a superfamily of heme-thiolate enzymes that catalyze the monooxygenation of inert substrates and are intimately involved in drug metabolism, biosynthesis of steroids, and detoxification of xenobiotics.1,2 Due to the ability of P450s to efficiently catalyze the monooxygenation of unactivated C−H bonds under mild conditions, they have garnered much attention as potential biocatalysts for environmentally friendly synthetic chemistry.3−8 Given that bacterial P450s are readily obtained in their soluble form by employing conventional Escherichia coli gene expression systems, and that they possess very high catalytic activities, these enzymes are regarded as promising candidates for the development of effective biocatalysts. However, their restricting substrate specificities result in trivial catalytic activities toward non-native substrates. To utilize bacterial P450s for non-native substrate oxidation, their substrate specificity needs to be altered, which has conventionally been achieved through mutagenesis approaches. P450s activate molecular oxygen at the heme iron, which is ligated to the thiolate of cysteine as the fifth ligand, to generate the active oxygen species (iron(IV)-oxo porphyrin π cation radical), so-called Compound I.9 The catalytic cycle of P450 can be broken down into following five steps (Figure 1): (1) Substrate binding, resulting in the removal of a water molecule ligated to the heme iron, causing a positive shift in redox potential of the heme iron;10 (2) first electron transfer, reduction of Fe3+ (ferric) to Fe2+ (ferrous) by electron transfer from NADPH through the reductase domain; (3) binding of molecular oxygen to ferrous heme; (4) second electron transfer yielding Compound 0, (5) O−O bond cleavage yielding Compound I; and (6) oxidation of the bound substrate by Compound I.1 According to this reaction mechanism, appropriate binding of the substrate to the active site of P450s is crucial for initiation of the catalytic cycle. Therefore, molecules with structures deviating from those of the native substrates cannot initiate the first step of the catalytic cycle. To provide appropriate space to accommodate non-native substrates, a plethora of mutant P450s, such as those of P450BM34 and P450cam,11,12 have been prepared by site-directed mutagenesis, as well as by random mutagenesis. The resulting mutants were

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CYTOCHROME P450BM3 CYP102A1 (P450BM3) isolated from Bacillus megaterium is a structurally self-sufficient P450 enzyme; that is, its reductase domain and P450 domain are fused together on a single peptide chain, and possesses some of the highest monooxygenase activities among all P450s reported to date.18 P450BM3 catalyzes the hydroxylation of fatty acids bearing alkyl chain lengths ranging from 12 to 20 (C12−20) carbon units and the turnover number for the hydroxylation of arachidonic acid (C20) has been reported to reach as high as 17 100 min−1 (kcat).18 Given its record catalytic activity, P450BM3 is anticipated to be suitable for the hydroxylation of various unactivated C−H bonds; however, its high substrate specificity poses an obstacle for use as a biocatalyst. It is presumed that the substrate recognition mechanism of P450BM3 dictates its high substrate specificity and understanding what governs substrate recognition is the key to overcoming this obstacle. The X-ray crystal structure of P450BM3 with palmitoleic acid reveals that palmitoleic acid is fixed by two major interactions (Figure 2): (i) Ionic interactions of the substrate carboxylate with Arg47 and Tyr51 of P450BM3, and (ii) hydrophobic interactions between the alkyl chain of the fatty acid with amino acids at the substrate binding site.19 Fatty acid binding to P450BM3 induces initiation B


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Figure 2. Overall structure (a) and active site structure (b) of P450BM3 (PDB ID: 1FAG).

Figure 3. Schematic representation of fatty acid hydroxylation (upper) and propane hydroxylation in the presence of a decoy molecule (bottom) catalyzed by P450BM3. Reaction of propane in the absence of any decoy molecule (middle).

molecules can initiate the activation of molecular oxygen to generate Compound I in the same fashion as long-chain fatty acids (Figure 3). Furthermore, we presumed that, due to the high bond strength of C−F bonds (C−F bond dissociation energy: 116 kcal/mol), PFCs are not oxidizable22 and Compound I can be utilized for the oxidation of non-native substrates. PFCs were envisioned to bind to the active site of P450BM3 through interaction of the PFCs carboxylic groups with Arg47 and Tyr51, reminiscent of the natural substrate. Shorter PFCs of 8−14 carbon atoms in length (PFC8−PFC14) were anticipated to provide sufficient space for non-native substrates, as the structure of the substrate-binding site of P450BM3 is capable of comfortably accommodating fatty acids bearing 16 carbon atoms (Palmitic acid). Non-native-substrates such as propane, butane, and cyclohexane were hydroxylated in the presence of PFCs, while in the absence of any PFC no product was detected. The rate of product formation and coupling efficiency for the hydroxylation of small alkanes in the presence of a series of PFCs are summarized in Table 1. These results demonstrated that the addition of a decoy molecule to wild-type P450BM3 is required to impart P450BM3 with the ability to catalyze the

of the catalytic cycle by removing a water molecule ligated to the heme iron (Figure 1). This causes a positive shift in redox potential of the heme iron,10 which is crucial for the generation of Compound I. Accordingly, non-native substrates, whose structures differ significantly from those of native substrates, cannot effectively initiate the first step of the catalytic cycle, prohibiting progression of catalysis.

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NON-NATIVE SUBSTRATE HYDROXYLATION EMPLOYING PERFLUOROCARBOXYLIC ACIDS (PFCs) AS DECOY MOLECULES We conceived that the straightforward addition of a series of PFCs as inert dummy substrates (decoy molecules) could transform wild-type P450BM3 into a small alkane hydroxylase, without the need to replace any amino acid residues.20,21 This strategy for the hydroxylation of non-native substrates is shown in Figure 3. Indeed, upon the addition of perfluorocarboxylic acids, as decoy molecules, consumption of NADPH was observed. This indicated that induction of the first step in the catalytic cycle, required for the formation of Compound I, was successful. Consequently, we can confidently assume that decoy C


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Table 1. Turnover Rate and Coupling Efficiency of Propane, Butane, Cyclohexane, and Benzene Hydroxylation Catalyzed by P450BM3 in the Presence of Perfluorinated Carboxylic Acids (PFCs) propane hydroxylation decoy molecule

rate [min−1 P450−1]

none PFC8 PFC9 PFC10 PFC11 PFC12 PFC13 PFC14

not detected not detected 24 ± 8 67 ± 2 40 ± 4 25 ± 6 11 ± 3 not detected

coupling efficiency [%]

13 18 9 7 3

butane hydroxylation

cyclohexane hydroxylation





not detected 2±3 110 ± 30 100 ± 30 69 ± 20 69 ± 15 35 ± 10 13 ± 3

1 55 17 11 10 4 2

not detected 63 ± 4 110 ± 10 72 ± 13 54 ± 10 44 ± 8 26 ± 1 16 ± 2

45 37 23 17 15 8 5

benzene hydroxylation rate [min−1 P450−1]


not detected 38 ± 6 120 ± 9 120 ± 5 83 ± 6 71 ± 5

17 24 19 15 12

of P450BM3 for non-native substrates remains much lower than for natural substrates. We presumed that the interaction of PFCs with P450BM3 is not sufficient to draw out the full potential of the enzyme. Previous reports show that modification of palmitic acid with glycine (N- palmitoylglycine) increases hydroxylation efficiency,26 which can to some extent be attributed to an increase in the binding strength of N- palmitoylglycine (Kd = 262 nM). Crystal structure analysis lends further support, revealing additional hydrogen-bonding interactions of the carboxylate moiety of N-palmitoylglycine with Gln73 and Ala74 of P450BM3 (Figure 4).26 These observations were translated into the development of a novel generation of decoy molecules, henceforth referred to as second-generation decoy molecules, where, analogous to N- palmitoylglycine, PFCs were modified with amino acids. Thus, a series of N-perfluoroacyl amino acids with hydrophobic side chains (Figure 5) were prepared, and the effects of amino acid side chain structure and chirality in combination with alkyl chain length of the decoy molecule on propane hydroxylation were examined (Table 2).27 Hydroxylation activities were drastically improved by the introduction of a single amino acid residue to the decoy molecule (Table 2), accompanied by improved binding affinity of the decoy molecule. For example, the Kd of PFC10 was estimated to be 290 μM, while the Kd of PFC10-Trp was 3 μM, which represents a 100 fold increase in binding strength. Among the examined Nperfluoropelargonoyl amino acids (PFC9-amino acids), N-

hydroxylation of gaseous alkanes. Interestingly, the rate of product formation was influenced significantly by the length of the PFC’s alkyl chain. PFC10 elicited the fastest rate of hydroxylation of propane (67 min−1 P450−1) and the highest coupling efficiency (18%) among examined PFCs. For butane hydroxylation, PFC9 and PFC10 were almost equally effective and displayed the fastest rate of sec-butanol formation, 100−113 min−1 P450−1. With cyclohexane, PFC9 elicited the fastest rate of cyclohexanol formation (110 min−1 P450−1), and a coupling efficiency of 35%. These results indicated that the larger alkanes tended to prefer PFCs with shorter alkyl-chains and that the efficiency of hydroxylation by P450BM3 is governed by a combination of PFC length and alkane size. Under high-pressure conditions of 0.5 MPa ethane with PFC10 as a decoy molecule even the primary carbon of ethane could be hydroxylated (40 h−1 P450−1), indicating that when the concentration of substrate in the reaction mixture is increased even primary carbons can be hydroxylated using the P450BM3−decoy molecule system.23 In addition to the hydroxylation of small alkanes, the hydroxylation of aromatics, such as benzene and monosubstituted benzenes, has also been achieved using the P450BM3− decoy molecule system (Table 1).24 For benzene hydroxylation, PFC9 and PFC10 afforded the fastest turnover rate of 120 min−1 P450−1. The coupling efficiency of 24%, for PFC9, was the highest among examined PFCs, indicating that the active site space provided by PFC9 is suitable for the accommodation of benzene. Of note, the catalytic turnover rate and coupling efficiency for the decoy molecule system exceeded those of engineered P450BM3 mutants prepared by directed evolution.25 According to high-performance liquid chromatography, selectivity for phenol production was more than 99% (data not shown) with no detectable overoxidation products. As the heme cavity of P450BM3 is lined with hydrophobic amino acid residues, phenol is likely rapidly expelled from the active site without consecutive rebinding, thus preventing subsequent overoxidation. Toluene was also hydroxylated and PFC9 yielded the fastest turnover rate of 220 min−1P450−1 for selective hydroxylation at the ortho-position. ortho-Selective hydroxylation was also observed with anisole, chlorobenzene, nitrobenzene, and acetophenone, showing that, regardless of the nature of the substituents, monosubstituted benzenes are specifically hydroxylated at the ortho-position.

Figure 4. Substrate-binding site of P450BM3 in complex with Npalmitoylglycine (NPG) (PDB ID: 1JPZ). No water molecule was coordinated to the heme iron.

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perfluoropelargonoyl-L-leucine (PFC9-Leu) proved the most effective for propane hydroxylation (product formation rate (PFR) = 256 min−1 P450−1; Table 2), which exceeded that of perfluorocapric acid (PFC10) (67 min−1 P450−1) by a factor of 4.20 Higher binding affinity of the decoy molecule may offer an explanation for the increased rates of hydroxylation. It is of interest to note here that decoy molecules containing the natural

SECOND GENERATION DECOY MOLECULES: N-PERFLUOROACYL AMINO ACIDS We have demonstrated the hydroxylation of non-native substrates by employing simple perfluorinated fatty acids as decoy molecules. Nevertheless, with PFCs the catalytic activity D


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Attempts to procure cocrystals of P450BM3 with perfluorocarboxylic acids (PFCs) bore no fruit, as electron density assignable to the decoy molecule could not be discerned. Fortunately, cocrystals of P450BM3 with PFC9-Trp revealed clear electron density in the fatty acid-binding channel that was readily assigned to PFC9-Trp (Figure 6a and b).27 Three distinct hydrogen-bonding interactions between PFC9-Trp and the amino acids Tyr51, Gln73, and Ala74 of P450BM3 could be identified (Figure 6b). One prominent difference between the decoy molecule-bound form and the native substrate-bound forms of P450BM3 is found for Arg47, which points away from the substrate toward the surface of the protein (Figures 4 and 6b), providing space for the accommodation of the hydrophobic amino acid side chain. Intriguingly, in place of an axial water molecule, typically found ligated to heme, a molecule of DMSO accommodated between Phe87 and Ala264 can be observed (Figure 6b−d). Crystal structure analysis also revealed why medium-chain-length decoy molecules were effective for the hydroxylation of small molecules, despite decoy molecules bearing longer alkyl chains binding more strongly to P450BM3. The crystal structure of P450BM3-bound PFC9-Trp indicated that the terminal perfluoromethyl group of PFC9-Trp is close to DMSO. This observation implies that, in the case of decoy molecules with alkyl chains longer than nine carbon units, either the binding site is occluded or access of gaseous alkanes is blocked by the alkyl chain of the decoy molecule. In both cases, lower catalytic activity and higher levels of uncoupling would be expected (Table 1).

Figure 5. Structures of second generation of decoy molecules. L-amino acid (PFC9-Ala) were preferred over the unnatural Denantiomer (PFC9-D-Ala; Table 2).

Table 2. Hydroxylation of Propane Catalyzed by Wild-Type P450BM3 with PFC-Amino Acida PF-amino acid

i-propanol PFR [min−1 P450−1]

n-propanol PFR [min−1 P450−1]


PFC6-Gly PFC7-Gly PFC8-Gly PFC9-Gly PFC10-Gly PFC11-Gly PFC9-DL-Ala PFC9-Ala PFC9-D-Ala PFC9-Val PFC9-Ile PFC8-Leu PFC9-Leu PFC10-Leu PFC11-Leu PFC9-Met PFC9-Phe PFC9-Trp

− − 60 ± 4 128 ± 17 89 ± 12 78 ± 27 124 ± 16 152 ± 11 111 ± 7 218 ± 41 228 ± 10 174 ± 13 256 ± 73 111 ± 20 48 ± 2 218 ± 12 251 ± 35 207 ± 22

− − 17 ± 4 31 ± 10 17 ± 2 15 ± 4 21 ± 1 20 ± 1 21 ± 1 23 ± 2 23 ± 3 19 ± 1 23 ± 1 28 ± 6 22 ± 4 20 ± 1 22 ± 2 20 ± 1

− − 17 33 21 17 21 23 23 29 26 33 36 16 9 34 40 34

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THIRD GENERATION DECOY MOLECULES: NONFLUORINATED N-ACYL AMINO ACIDS We succeeded in enhancing the catalytic activity of P450BM3 toward gaseous alkanes by developing a series of secondgeneration decoy molecules and in solving the crystal structure of P450BM3 in complex with N-perfluoropelargonoyl-Ltryptophan (PFC9-Trp) (PDB ID: 3WSP). From the crystal structure, it became clear that the terminus of the decoy molecule’s alkyl chain is fixed tightly through multiple hydrogenbonding interactions between the carboxyl and carbonyl groups

a

PFR = product formation rate.

Figure 6. Overall structure (a) and active site structure (b) of P450BM3 in complex with PFC9-Trp (PDB ID: 3WSP). Top view (c) and side view (d) of the active site structure shown as spheres. E


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Phe and the heme iron was measured at 8.4 Å (Figure 8). It is worth noting that the proline side chain of Z-Pro-Phe occupied the space adjacent to Pro25 (Figure 8). We presume that the bulkiness of proline at the center of Z-Pro-Phe may play a crucial role in positioning Z-Pro-Phe at the entrance of the fatty acid binding channel to create adequate space at the distal side of heme for benzene binding. We also employed structurally diverse third-generation decoy molecules to control the stereoselectivity of hydroxylation, yielding clear inversion of stereoselectivity for the benzylic hydroxylation of indane. When 5-cyclohexylvaleroyl-L-phenylalanine (5CHVA-Phe) was employed as the decoy molecule, the hydroxylation of Indane

of PFC9-Trp and the amino acids Tyr51, Gln73, and Ala74 of P450BM3. These interactions hold the decoy molecule firmly in place, preventing any slippage of the decoy further into the active site, thereby positioning the terminus at a secure distance from the active site heme iron (Figure 6b). In light of these findings, we conjectured that the alkyl chain terminus of nonfluorinated pelargonic acid (C9) modified with tryptophan (C9-Trp) would be bound sufficiently distant from the heme iron, preventing any undesired hydroxylation by P450BM3 (Figure 7). Indeed, Nacyl amino acids, such as pelargonoyl-L-phenylalanine (C9Phe), can serve as decoy molecules and, unexpectedly, benzene was more efficiently hydroxylated in the presence of N-acyl amino acids than their perfluorinated counterparts, indicating that fluorination is not necessarily required for decoy molecule function.28 Similar to second-generation decoy molecules, the catalytic turnover rate of third-generation decoy molecules was highly dependent upon the type of amino acid modification as well as the alkyl chain length. Pelargonic acid derivatives modified with L-amino acids bearing bulkier side chains exhibited a tendency for higher catalytic activities. Of all C9-Lamino acids examined, C9-Phe afforded the highest turnover rate (192 min−1 P450−1) for the hydroxylation of benzene (Figure 7). Alleviating the requirement for perfluorination, diverse carboxylic acids including dipeptides could now be

Figure 8. Substrate-binding site of P450BM3 in complex with Z-ProPhe (PDB ID: 5XA3). The DMSO molecule coordinated to the heme iron is not shown.

afforded 1-indanol with 53% (R) ee. In contrast, Z-Pro-Phe yielded 1-indanol with 56% (S) ee, indicating that decoy molecules can alter stereoselectivity (Figure 9).29

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HEME REPLACEMENT OF CYTOCHROME P450BM3 AND REACTIVITY OF Mn-SUBSTITUTED P450BM3 We successfully prepared the heme domain of P450BM3 without its heme (apo form) and applied it to the construction of metal-substituted P450BM3 (Figure 10).30 Our method is exceptionally simple and requires neither harsh conditions, e.g. acid/organic solvents, nor special heme transportation systems. Apo-P450BM3 was prepared by expressing the protein in ironlimiting M9 medium, where the biosynthesis of heme is suppressed. Reconstitution of metal-substituted P450BM3 heme domain was achieved during the course of cell homogenization via effortless addition of the target metal complex into the cell suspension without prior purification of apo protein. Manganese protoporphyrin IX (Mn-PPIX) was used as the metal complex and purified P450BM3 heme domain containing Mn-PPIX (Mn−P450BM3 heme domain) yielded a UV−Vis spectrum hinting at Mn-PPIX being incorporated at an appropriate position in the enzyme’s heme-binding pocket. Further support was lent by inductively coupled plasma optical emission spectrometry (ICP-OES), where only negligible amounts of iron could be detected. Decisive evidence for the correct incorporation of Mn-PPIX was obtained by X-ray crystallographic analysis of Mn−P450BM3 (PDB ID: 5ZIS), which revealed the overall structure to be almost identical to that of Fe−P450BM3. Moreover, from an anomalous difference Fourier map a significant peak at the center of protoporphyrin IX, indicating the presence of a manganese atom, could be observed. It should be noted that this reconstitution approach is

Figure 7. Structures of third generation decoy molecules and turnover frequencies of benzene hydroxylation.

exploited as building blocks, and a library of decoy molecules possessing diverse structures was prepared. Among the examined third-generation decoy molecules, N-enanthyl-Lproline modified with L-phenylalanine (C7-Pro-Phe) afforded the maximum turnover rate for benzene hydroxylation of 259 min−1 P450−1 with a coupling efficiency of 43% (Figure 7). The total turnover number (TON) for a 12 h reaction reached 40200 with a coupling efficiency of 46%. The crystal structure of the ZL-prolyl-L-phenylalanine (Z-Pro-Phe) bound to P450BM3 was solved at a resolution of 2.2 Å (Figure 8; PDB ID: 5XA3), where the typical hydrogen-bonding interactions with the amino acids Tyr51, Gln73, and Ala74 could be observed. The distance between the terminal carbon atom of the phenyl group of Z-ProF


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this obstacle, we developed a novel methodology, wherein the reconstituted heme domain is ligated to the reductase domain to procure reconstituted full length enzyme (Figure 10). The transpeptidase sortase A (SrtA) was chosen to connect the heme domain to the reductase domain.34 SrtA can recognize short amino acid sequences, LPXTG (X can be any amino acid) and oligoglycine (two or more glycines), and will indiscriminately form a new peptide bond (LPXTGG...) if the required sequence motif is present. After optimization of the linker region between the P450BM3 heme and reductase domain, we prepared constructs of the P450BM3 heme domain with LPATGG at the C-terminus (1-456-LPATGG) and GG at the N-terminus of the reductase domain (GG-458-1049). SrtA-mediated ligation was accomplished by simply mixing the heme domain, the reductase domain, and SrtA together. Ligated full-length P450BM3 exhibited almost the same catalytic activity as wildtype P450BM3. After demonstrating that this method works with nonreconstituted P450BM3, we utilized our technique to prepare Mn-substituted full-length P450BM3 (Mn−P450BM3) and applied it to the hydroxylation of propane. When PFC9-Trp was employed as the decoy molecule Mn−P450BM3 successfully yielded propanol as a product, but, more interestingly, the product selectivity diverged from that of Fe−P450BM3. With Fe−P450BM3 the main product was i-propanol, due to the C− H bond dissociation energy of primary carbons being higher than that of secondary carbon atoms. However, in the case of Mn−P450BM3, the product contained more n-propanol, with the ratio of n-propanol to i-propanol being 17.5%, compared to 4.8% for Fe−P450BM3. Although the active species of Mn− P450BM3 has not yet been determined, we strongly believe that it can efficiently hydroxylate the more challenging primary carbon of propane and may be effective for the hydroxylation of demanding substrates. This represents a good example of how the reactive properties of P450BM3 can be tweaked by substituting its catalytic center and how heme-substitution could potentially expand the research field of P450BM3.

Figure 9. Hydroxylation of indane catalyzed by P450BM3 in the presence of Z-Pro-Phe and 5CHVA-Phe.

also applicable to other hemoproteins and we have succeeded in the reconstitution of H-NOXs with Mn-PPIX and ruthenium mesoporphyrin IX (Ru-MPIX). Recently, the Hartwig and Fasan groups also applied iron-limiting media to obtain various metal-substituted hemoproteins and even utilized them for carbene C−H insertion reactions.31,32 Having established a functioning protocol for the preparation of the reconstituted P450BM3 heme domain, we set out to develop a novel methodology for the reconstitution of fulllength P450BM3, as there have been no reports on the heme substitution of P450BM3 in its full-length form until our recent report.33 Expression of full-length P450BM3 in nutrient-limiting medium is thought to potentially cause irreversible denaturation as well as loss of other cofactors, rendering the aforementioned heme-reconstitution methodology unsuitable. P450BM3 is fused to its redox partner (FAD and FMN domains) and this unique structural feature aids the efficient electron transfer from NADPH to heme, which is essential for the generation of Compound I. Accordingly, the heme domain by itself is insufficient for effective oxidation, even when heme is substituted by potent metal complexes. In order to overcome

Figure 10. Reconstitution of heme-substituted full-length P450BM3 with a synthetic metal complex by the SrtA-mediated protein−protein ligation based on the heme substitution methodology of P450BM3 heme domain under mild conditions. (a) Reconstitution of heme domain of P450BM3 at the disruption stage of Escherichia coli cells. (b) SrtA-mediated ligation of the heme domain of P450BM3 and reductase domain. G


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Figure 11. Whole-cell biocatalyst for benzene hydroxylation activated by decoy molecules.

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ACTIVATION OF INTRACELLULAR P450BM3 BY THIRD GENERATION DECOY MOLECULES One significant drawback, hindering further in vitro application of P450BM3 with decoy molecules and in general, is that a stoichiometric amount of expensive cofactors, NADPH, are required as electron donors to drive the reaction. An attractive solution to overcome this limitation is the development of whole-cell reaction systems, wherein NADPH is produced and regenerated cost-efficiently by the bacteria.4 To expand the scope of the P450BM3-decoy molecule system for practical application in synthetic chemistry, a facile whole-cell biocatalysis system for the direct hydroxylation of benzene was examined, wherein wild-type P450BM3 expressed in E. coli could be activated by the straightforward addition of decoy molecules to the culture medium. We hypothesized that decoy molecules should be able to traverse the outer membrane of E. coli cells, thereby activating intracellular P450BM3 for the hydroxylation of non-native substrates (Figure 11). E. coli cells expressing P450BM3 were suspended in a phosphate buffer containing 10 mM benzene and 100 μM decoy molecules, and incubated for 5 h at room temperature. Unsurprisingly, in the absence of any decoy molecule, P450BM3 barely catalyzed the transformation of benzene to phenol (Table 3). Noteworthy, the formation of phenol varied drastically, depending upon the structure of the decoy molecule. Among a series of third-generation decoy molecules (Table 3), (S)-Ibuprophen modified with L-phenylalanine ((S)-Ibu-Phe) and N-enanthyl-L-prolyl-L-phenylalanine (C7-Pro-Phe) evoked the highest phenol yield (38%).35 Furthermore, the dependence of decoy molecule concentration upon the hydroxylation activity was examined and C7-Pro-Phe was found to remain effective even at very low concentrations, with 5 μM being sufficient to elicit the maximum catalytic activity, 44% yield for a 12 h reaction. Intriguingly, although (S)Ibu-Phe and C7-Pro-Phe yielded comparable turn overs in vitro, C7-Pro-Phe was effective in vivo at concentrations as low as 5 μM, potentially due to superior cell membrane permeability, making it superior for application in vivo. Under optimized conditions, using OD600 = 12.6 of whole-cell biocatalyst, the phenol yield reached 59% with 75% of benzene being converted to either phenol or hydroquinone (phenol selectivity: 78%).35

Table 3. Product Formations and Yields of Whole-Cell Benzene Hydroxylationa decoy molecule

phenol conc. [μM]

GC yield [%]

none PFC9 PFC9-Phe C9-Trp C9-Phe C10-Phe C11-Phe (R)-Ibu-Phe (S)-Ibu-Phe Z-Pro-Phe C7-Pro-Phe

40 ± 10 60 ± 3 190 ± 30 170 ± 20 790 ± 80 920 ± 50 440 ± 20 1960 ± 20 3760 ± 110 300 ± 50 3810 ± 150

0.4 0.6 1.9 1.7 7.9 9.2 4.4 20 38 3.0 38

a

Reaction conditions: E. coli BL21(DE3) expressing wild-type P450BM3 (OD600 = 6.3), decoy molecule (100 μM), benzene (10 mM), glucose (40 mM), at 25 °C for 5 h in 110 mM phosphate buffer, pH 7.4.

addition of decoy molecules that induce substrate misrecognition. Decoy molecules alleviate the high substrate specificity of P450BM3, thereby accelerating the generation of the active species, and enabling the oxidation of non-native substrates, without the requirement for mutagenesis. The catalytic activity is clearly dependent upon the structure of the decoy molecule, implying that the oxidation of non-native substrates by P450BM3 could be enhanced even further by fine-tuning of second- and third-generation decoy molecule structures. For example, the enantioselectivity of non-native substrate hydroxylation could be inverted by tweaking the structure of decoy molecules. Moreover, the regioselectivity of propane hydroxylation by P450BM3 could be influenced by changing the heme metal from iron to manganese. Decoy molecules were shown to be able to permeate into the cell, permitting activation of intracellular P450BM3 expressed in E. coli, enabling the successful construction of a whole-cell bioreactor for the biotransformation of benzene to phenol without the need for exogenously added costly NADPH. This represents a novel reaction concept, wherein external additives bestow a new catalytic function upon the enzyme in vivo. Recently, it was also reported that decoy molecules can be applied to other biotransformations.36,37 We believe that the principle of the decoy molecule system holds the potential to be transferred to many more enzymatic reactions. Furthermore, a combination of

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CONCLUSION In this Account, we have established that P450BM3 can be fooled into oxidizing non-native substrates via the simple H


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JP15H05806 in Precisely Designed Catalysts with Customized Scaffolding to O.S.

mutagenesis with the decoy molecule system may open the door for solving limitations imposed by the substrate specificity of enzymatic reactions.

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AUTHOR INFORMATION

REFERENCES

(1) Ortiz de Montellano, P. R.: Cytochrome P450 Structure, Mechanism, and Biochemistry; Springer US, 2005. (2) Sono, M.; Roach, M. P.; Coulter, E. D.; Dawson, J. H. Hemecontaining oxygenases. Chem. Rev. 1996, 96, 2841−2887. (3) Denisov, I. G.; Makris, T. M.; Sligar, S. G.; Schlichting, I. Structure and chemistry of cytochrome P450. Chem. Rev. 2005, 105, 2253−2277. (4) Whitehouse, C. J. C.; Bell, S. G.; Wong, L.-L. P450(BM3) (CYP102A1): connecting the dots. Chem. Soc. Rev. 2012, 41, 1218− 1260. (5) Fasan, R. Tuning P450 Enzymes as Oxidation Catalysts. ACS Catal. 2012, 2, 647−666. (6) Grogan, G. Cytochromes P450: exploiting diversity and enabling application as biocatalysts. Curr. Opin. Chem. Biol. 2011, 15, 241−248. (7) O’Reilly, E.; Koehler, V.; Flitsch, S. L.; Turner, N. J. Cytochromes P450 as useful biocatalysts: addressing the limitations. Chem. Commun. 2011, 47, 2490−2501. (8) Schulz, S.; Girhard, M.; Urlacher, V. B. Biocatalysis: Key to Selective Oxidations. ChemCatChem 2012, 4, 1889−1895. (9) Rittle, J.; Green, M. T. Cytochrome P450 Compound I: Capture, Characterization, and C-H Bond Activation Kinetics. Science 2010, 330, 933−937. (10) Daff, S. N.; Chapman, S. K.; Holt, R. A.; Govindaraj, S.; Poulos, T. L.; Munro, A. W. Redox control of the catalytic cycle of flavocytochrome P-450 BM3. Biochemistry 1997, 36, 13816−13823. (11) Bell, S. G.; Stevenson, J. A.; Boyd, H. D.; Campbell, S.; Riddle, A. D.; Orton, E. L.; Wong, L. L. Butane and propane oxidation by engineered cytochrome P450(cam). Chem. Commun. 2002, 490−491. (12) Bell, S. G.; Orton, E.; Boyd, H.; Stevenson, J. A.; Riddle, A.; Campbell, S.; Wong, L. L. Engineering cytochrome P450cam into an alkane hydroxylase. Dalton Transactions 2003, 2133−2140. (13) Fasan, R.; Chen, M. M.; Crook, N. C.; Arnold, F. H. Engineered alkane-hydroxylating cytochrome P450(BM3) exhibiting nativelike catalytic properties. Angew. Chem., Int. Ed. 2007, 46, 8414−8418. (14) Fasan, R.; Meharenna, Y. T.; Snow, C. D.; Poulos, T. L.; Arnold, F. H. Evolutionary History of a Specialized P450 Propane Monooxygenase. J. Mol. Biol. 2008, 383, 1069−1080. (15) Chen, M. M. Y.; Snow, C. D.; Vizcarra, C. L.; Mayo, S. L.; Arnold, F. H. Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 BM3-catalyzed hydroxylation of small alkanes. Protein Eng., Des. Sel. 2012, 25, 171−178. (16) Meinhold, P.; Peters, M. W.; Chen, M. M. Y.; Takahashi, K.; Arnold, F. H. Direct conversion of ethane to ethanol by engineered cytochrome P450BM3. ChemBioChem 2005, 6, 1765−1768. (17) Xu, F.; Bell, S. G.; Lednik, J.; Insley, A.; Rao, Z. H.; Wong, L. L. The heme monooxygenase cytochrome P450(cam) can be engineered to oxidize ethane to ethanol. Angew. Chem., Int. Ed. 2005, 44, 4029− 4032. (18) Girvan, H. M.; Marshall, K. R.; Lawson, R. J.; Leys, D.; Joyce, M. G.; Clarkson, J.; Smith, W. E.; Cheesman, M. R.; Munro, A. W. Flavocytochrome P450BM3 mutant A264E undergoes substratedependent formation of a novel heme iron ligand set. J. Biol. Chem. 2004, 279, 23274−23286. (19) Li, H. Y.; Poulos, T. L. The structure of the cytochrome p450BM3 haem domain complexed with the fatty acid substrate, palmitoleic acid. Nat. Struct. Biol. 1997, 4, 140−146. (20) Kawakami, N.; Shoji, O.; Watanabe, Y. Use of Perfluorocarboxylic Acids To Trick Cytochrome P450BM3 into Initiating the Hydroxylation of Gaseous Alkanes. Angew. Chem., Int. Ed. 2011, 50, 5315−5318. (21) Zilly, F. E.; Acevedo, J. P.; Augustyniak, W.; Deege, A.; Haeusig, U. W.; Reetz, M. T. Tuning a P450 Enzyme for Methane Oxidation. Angew. Chem., Int. Ed. 2011, 50, 2720−2724. (22) Banks, R. E.; Tatlow, J. C. A guide to modern organofluorine chemistry. J. Fluorine Chem. 1986, 33, 227−346.

Corresponding Author

*(O.S.) Fax: (+81)52-789-3557. E-mail: [email protected]. nagoya-u.ac.jp. ORCID

Osami Shoji: 0000-0003-3522-0065 Notes

The authors declare no competing financial interest. Biographies Osami Shoji received his B.S. (1997), M.S. (1999), and Ph.D. (2002) degrees in polymer chemistry from Chiba University under the direction of Prof. Takayuki Nakahira. He joined Prof. Yoshiaki Kobuke’s group at the Nara Institute of Science and Technology as a postdoctoral fellow. In 2005, he joined Prof. Yoshihito Watanabe’s group as a postdoctoral fellow at the Graduate School of Science, Nagoya University. He was appointed as a JSPS postdoctoral fellow in 2006. In 2008, he was promoted to the position of assistant professor at the Graduate School of Science, Nagoya University. He has been an associate professor since 2013. His research interests include construction and characterization of biocatalysts. Yuichiro Aiba obtained his B.S., M.S., and PhD degrees from the University of Tokyo in 2005, 2007, and 2010, respectively, under the supervision of Prof. Makoto Komiyama. During this period, he was also a JSPS research fellow (DC1; 2007−2010). In 2010, he was appointed as a specially appointed assistant professor at the University of Tokyo. In 2012, he joined Prof. David R. Corey’s group as a JSPS postdoctoral fellow for research abroad at the Southwestern Medical Center, University of Texas. Since 2015, he has been an assistant professor at the Graduate School of Science, Nagoya University. His research interests include regulation of DNA and RNA function by artificial nucleic acid, peptide chemistry, and construction of biocatalysts based on protein engineering. Yoshihito Watanabe received his B.Sc. degree from Tohoku University and a Ph.D. degree under the direction of Prof. Shigeru Oae of Tsukuba University in 1982. He joined Prof. John T. Groves’ group at the University of Michigan as a postdoctoral fellow then moved to Princeton University as a research staff member in 1985. In 1987, he was appointed as an assistant professor in the Department of Biochemistry, Medical School of Keio University, Japan. In 1989, he joined the National Chemical Research Laboratories in Tsukuba as a senior research scientist. He was appointed as an associate professor at Kyoto University in 1990. From 1994 he spent 8 years at the Institute for Molecular Science as a professor and then joined the current faculty at Nagoya University as a full professor in 2002. In 2009, he was appointed as Vice President in charge of International Affairs. His research interests include the molecular mechanisms of oxygen activation by heme enzymes and the molecular design of metalloenzymes.

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ACKNOWLEDGMENTS This work was supported by Grants-in-Aid for Scientific Research (S) to Y.W. (24225004) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan) and JST CREST Grant Number JPMJCR15P3, Japan. This work was also supported by JSPS KAKENHI Grant Number I


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Accounts of Chemical Research (23) Kawakami, N.; Shoji, O.; Watanabe, Y. Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules. Chem. Sci. 2013, 4, 2344−2348. (24) Shoji, O.; Kunimatsu, T.; Kawakami, N.; Watanabe, Y. Highly Selective Hydroxylation of Benzene to Phenol by Wild-type Cytochrome P450BM3 Assisted by Decoy Molecules. Angew. Chem., Int. Ed. 2013, 52, 6606−6610. (25) Farinas, E. T.; Alcalde, M.; Arnold, F. Alkene epoxidation catalyzed by cytochrome P450BM-3 139−3. Tetrahedron 2004, 60, 525−528. (26) Haines, D. C.; Tomchick, D. R.; Machius, M.; Peterson, J. A. Pivotal role of water in the mechanism of P450BM-3. Biochemistry 2001, 40, 13456−13465. (27) Cong, Z.; Shoji, O.; Kasai, C.; Kawakami, N.; Sugimoto, H.; Shiro, Y.; Watanabe, Y. Activation of Wild-Type Cytochrorne P450BM3 by the Next Generation of Decoy Molecules: Enhanced Hydroxylation of Gaseous Alkanes and Crystallographic Evidence. ACS Catal. 2015, 5, 150−156. (28) Shoji, O.; Yanagisawa, S.; Stanfield, J. K.; Suzuki, K.; Cong, Z.; Sugimoto, H.; Shiro, Y.; Watanabe, Y. Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives. Angew. Chem., Int. Ed. 2017, 56, 10324−10329. (29) Suzuki, K.; Stanfield, J. K.; Shoji, O.; Yanagisawa, S.; Sugimoto, H.; Shiro, Y.; Watanabe, Y. Control of stereoselectivity of benzylic hydroxylation catalysed by wild-type cytochrome P450BM3 using decoy molecules. Catal. Sci. Technol. 2017, 7, 3332−3338. (30) Kawakami, N.; Shoji, O.; Watanabe, Y. Single-Step Reconstitution of Apo-Hemoproteins at the Disruption Stage of Escherichia coli Cells. ChemBioChem 2012, 13, 2045−2047. (31) Sreenilayam, G.; Moore, E. J.; Steck, V.; Fasan, R. Metal Substitution Modulates the Reactivity and Extends the Reaction Scope of Myoglobin Carbene Transfer Catalysts. Adv. Synth. Catal. 2017, 359, 2076−2089. (32) Key, H. M.; Dydio, P.; Clark, D. S.; Hartwig, J. F. Abiological catalysis by artificial haem proteins containing noble metals in place of iron. Nature 2016, 534, 534−537. (33) Omura, K.; Aiba, Y.; Onoda, H.; Stanfield, J. K.; Ariyasu, S.; Sugimoto, H.; Shiro, Y.; Shoji, O.; Watanabe, Y. Reconstitution of fulllength P450BM3 with an artificial metal complex by utilising the transpeptidase Sortase A. Chem. Commun. 2018, 54, 7892−7895. (34) Mazmanian, S. K.; Liu, G.; Ton-That, H.; Schneewind, O. Staphylococcus aureus Sortase, an Enzyme that Anchors Surface Proteins to the Cell Wall. Science 1999, 285, 760−763. (35) Karasawa, M.; Stanfield, J. K.; Yanagisawa, S.; Shoji, O.; Watanabe, Y. Whole-Cell Biotransformation of Benzene to Phenol Catalysed by Intracellular Cytochrome P450BM3 Activated by External Additives. Angew. Chem., Int. Ed. 2018, 57, 12264−12269. (36) Demming, R. M.; Hammer, S. C.; Nestl, B. M.; Gergel, S.; Fademrecht, S.; Pleiss, J.; Hauer, B. Asymmetric Enzymatic Hydration of Unactivated, Aliphatic Alkenes. Angew. Chem., Int. Ed. 2019, 0, 173. (37) Demming, R. M.; Otte, K. B.; Nestl, B. M.; Hauer, B. Optimized Reaction Conditions Enable the Hydration of Non-natural Substrates by the Oleate Hydratase from Elizabethkingia meningoseptica. ChemCatChem 2017, 9, 758−766.

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Hoodwinking Cytochrome P450BM3 into Hydroxylating Non-Native

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