Osami Shoji - Cochemist

Innenrücktitelbild: Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group (Angew. Chem. 48/2017)

Co-reporter:Hiromu Uehara;Yuma Shisaka;Tsubasa Nishimura;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro;Dr. Yoshihiro Miyake; Dr. Hiroshi Shinokubo; Dr. Yoshihito Watanabe;Dr. Osami Shoji

Angewandte Chemie 2017 Volume 129(Issue 48) pp:15675-15675

Publication Date(Web):2017/11/27

DOI:10.1002/ange.201710879

Das Hämbindeprotein HasA aus P. aeruginosa kann Eisen(III)-5,15-diphenylporphyrin und mehrere Derivate aufnehmen, auch wenn sie sperrige Substituenten in der meso-Position des Porphyrins tragen. O. Shoji et al. präsentieren in ihrer Zuschrift auf S. 15481 Kristallstrukturen dieser künstlichen HasAs und weisen nach, dass die wachstumsinhibierende Wirkung gegen P. aeruginosa je nach Struktur des eingefangenen Metallkomplexes variiert.

Frontispiece: Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Co-reporter:Dr. Osami Shoji;Sota Yanagisawa;Joshua Kyle Stanfield;Kazuto Suzuki;Dr. Zhiqi Cong;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro; Dr. Yoshihito Watanabe

Angewandte Chemie International Edition 2017 Volume 56(Issue 35) pp:

Publication Date(Web):2017/08/21

DOI:10.1002/anie.201783561

Hydroxylation In their Communication on page 10324 ff., O. Shoji, Y. Watanabe et al. show that certain amino acid derivatives efficiently activate cytochrome P450BM3 for the direct hydroxylation of benzene to phenol.

Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Co-reporter:Dr. Osami Shoji;Sota Yanagisawa;Joshua Kyle Stanfield;Kazuto Suzuki;Dr. Zhiqi Cong;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro; Dr. Yoshihito Watanabe

Angewandte Chemie International Edition 2017 Volume 56(Issue 35) pp:10324-10329

Publication Date(Web):2017/08/21

DOI:10.1002/anie.201703461

AbstractThe selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min−1 P450BM3−1 and 40 200 P450BM3−1 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

Inside Back Cover: Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group (Angew. Chem. Int. Ed. 48/2017)

Co-reporter:Hiromu Uehara;Yuma Shisaka;Tsubasa Nishimura;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro;Dr. Yoshihiro Miyake; Dr. Hiroshi Shinokubo; Dr. Yoshihito Watanabe;Dr. Osami Shoji

Angewandte Chemie International Edition 2017 Volume 56(Issue 48) pp:15471-15471

Publication Date(Web):2017/11/27

DOI:10.1002/anie.201710879

The heme acquisition protein HasA from P. aeruginosa can accommodate iron(III)-5,15-diphenylporphyrin and several derivatives despite the fact that they have bulky substituents at the meso position of the porphyrin. In their Communication on page 15279 ff., O. Shoji and co-workers present crystal structures of these artificial HasAs, and show that the growth inhibition effect against P. aeruginosa differs depending on the structure of the metal complex captured.

Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group

Co-reporter:Hiromu Uehara;Yuma Shisaka;Tsubasa Nishimura;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro;Dr. Yoshihiro Miyake; Dr. Hiroshi Shinokubo; Dr. Yoshihito Watanabe;Dr. Osami Shoji

Angewandte Chemie 2017 Volume 129(Issue 48) pp:15481-15485

Publication Date(Web):2017/11/27

DOI:10.1002/ange.201707212

AbstractIron(III)-5,15-diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa, despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron-limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.

Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Co-reporter:Dr. Osami Shoji;Sota Yanagisawa;Joshua Kyle Stanfield;Kazuto Suzuki;Dr. Zhiqi Cong;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro; Dr. Yoshihito Watanabe

Angewandte Chemie 2017 Volume 129(Issue 35) pp:10460-10465

Publication Date(Web):2017/08/21

DOI:10.1002/ange.201703461

AbstractThe selective hydroxylation of benzene to phenol, without the formation of side products resulting from overoxidation, is catalyzed by cytochrome P450BM3 with the assistance of amino acid derivatives as decoy molecules. The catalytic turnover rate and the total turnover number reached 259 min−1 P450BM3−1 and 40 200 P450BM3−1 when N-heptyl-l-proline modified with l-phenylalanine (C7-l-Pro-l-Phe) was used as the decoy molecule. This work shows that amino acid derivatives with a totally different structure from fatty acids can be used as decoy molecules for aromatic hydroxylation by wild-type P450BM3. This method for non-native substrate hydroxylation by wild-type P450BM3 has the potential to expand the utility of P450BM3 for biotransformations.

Frontispiz: Direct Hydroxylation of Benzene to Phenol by Cytochrome P450BM3 Triggered by Amino Acid Derivatives

Co-reporter:Dr. Osami Shoji;Sota Yanagisawa;Joshua Kyle Stanfield;Kazuto Suzuki;Dr. Zhiqi Cong;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro; Dr. Yoshihito Watanabe

Angewandte Chemie 2017 Volume 129(Issue 35) pp:

Publication Date(Web):2017/08/21

DOI:10.1002/ange.201783561

Hydroxylierung In der Zuschrift auf S. 10460 beschreiben O. Shoji, Y. Watanabe et al. die Verwendung von bestimmten Aminosäurederivaten für die Aktivierung von Cytochrom P450BM3 zur direkten Hydroxylierung von Benzol zu Phenol.

Use of apomyoglobin to gently remove heme from a H2O2-dependent cytochrome P450 and allow its reconstitution

Co-reporter:Shih-Cheng Chien;Yoshiko Morimoto;Yoshihito Watanabe

New Journal of Chemistry (1998-Present) 2017 vol. 41(Issue 1) pp:302-307

Publication Date(Web):2016/12/19

DOI:10.1039/C6NJ02882A

The heme of hydrogen peroxide-dependent cytochrome P450BSβ (P450BSβ) was removed by apomyoglobin under mild conditions to give apo-P450BSβ without the need for acidic conditions and organic solvents. The circular dichroism spectrum of the apo-P450BSβ was essentially identical to that of holo-P450BSβ, showing a small structural change resulting from the removal of heme using apomyoglobin. The apo-P450BSβ was reconstituted with hemin or manganese protoporphyrin IX (MnPPIX), and the resulting reconstituted P450BSβ catalyzed the one-electron oxidation of guaiacol using hydrogen peroxide as an oxidant. A higher catalytic activity was observed for P450BSβ reconstituted with MnPPIX when meta-chloroperoxybenzoic acid (mCPBA) was used as the oxidant.

Control of stereoselectivity of benzylic hydroxylation catalysed by wild-type cytochrome P450BM3 using decoy molecules

Co-reporter:Kazuto Suzuki;Joshua Kyle Stanfield;Sota Yanagisawa;Hiroshi Sugimoto;Yoshitsugu Shiro;Yoshihito Watanabe

Catalysis Science & Technology (2011-Present) 2017 vol. 7(Issue 15) pp:3332-3338

Publication Date(Web):2017/07/31

DOI:10.1039/C7CY01130J

The hydroxylation of non-native substrates catalysed by wild-type P450BM3 is reported, wherein “decoy molecules”, i.e., native substrate mimics, controlled the stereoselectivity of hydroxylation reactions. We employed decoy molecules with diverse structures, resulting in either a significant improvement in enantioselectivity or clear inversion of stereoselectivity in the benzylic hydroxylation of alkylbenzenes and cycloalkylbenzenes. For example, supplementation of wild-type P450BM3 with 5-cyclohexylvaleric acid-L-phenylalanine (5CHVA-Phe) and Z-proline-L-phenylalanine yielded 53% (R) ee and 56% (S) ee for indane hydroxylation, respectively, although 16% (S) ee was still observed in the absence of any additives. Moreover, we performed a successful crystal structure analysis of 5CHVA-L-tryptophan-bound P450BM3 at 2.00 Å, which suggests that the changes in selectivity observed were caused by conformational changes in the enzyme induced by binding of the decoy molecules.

Structures of the Heme Acquisition Protein HasA with Iron(III)-5,15-Diphenylporphyrin and Derivatives Thereof as an Artificial Prosthetic Group

Co-reporter:Hiromu Uehara;Yuma Shisaka;Tsubasa Nishimura;Dr. Hiroshi Sugimoto; Dr. Yoshitsugu Shiro;Dr. Yoshihiro Miyake; Dr. Hiroshi Shinokubo; Dr. Yoshihito Watanabe;Dr. Osami Shoji

Angewandte Chemie International Edition 2017 Volume 56(Issue 48) pp:15279-15283

Publication Date(Web):2017/11/27

DOI:10.1002/anie.201707212

AbstractIron(III)-5,15-diphenylporphyrin and several derivatives were accommodated by HasA, a heme acquisition protein secreted by Pseudomonas aeruginosa, despite possessing bulky substituents at the meso position of the porphyrin. Crystal structure analysis revealed that the two phenyl groups at the meso positions of porphyrin extend outside HasA. It was shown that the growth of P. aeruginosa was inhibited in the presence of HasA coordinating the synthetic porphyrins under iron-limiting conditions, and that the structure of the synthetic porphyrins greatly affects the inhibition efficiency.

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

Co-reporter:Osami Shoji, Takashi Fujishiro, Kousuke Nishio, Yukiko Kano, Hiroshi Kimoto, Shih-Cheng Chien, Hiroki Onoda, Atsushi Muramatsu, Shota Tanaka, Ayumi Hori, Hiroshi Sugimoto, Yoshitsugu Shiro and Yoshihito Watanabe

Catalysis Science & Technology 2016 vol. 6(Issue 15) pp:5806-5811

Publication Date(Web):22 Apr 2016

DOI:10.1039/C6CY00630B

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 that is able to catalyze monooxygenation of non-native substrates was constructed by one-point mutagenesis of P450SPα (CYP152B1). P450SPα, a long-alkyl-chain fatty acid hydroxylase, lacks any general acid–base residue around the heme. The carboxylate group of a fatty acid is thus indispensable for the generation of active species using H2O2. We prepared an A245E mutant to mimic a substrate-binding state by placing a carboxylate group at the active site. The active site structure of the A245E mutant is similar to that of the fatty-acid-bound state of P450SPα and catalyzes styrene oxidation at a rate of 280 min−1 (kcat), whereas the wild-type enzyme does not show any catalytic activity. More importantly, the same mutation, i.e. the mutation of the highly conserved threonine in P450s to glutamic acid, was also effective in introducing peroxygenase activity into P450BM3, P450cam, and CYP119. These results indicate that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.

Activation of Wild-Type Cytochrome P450BM3 by the Next Generation of Decoy Molecules: Enhanced Hydroxylation of Gaseous Alkanes and Crystallographic Evidence

Co-reporter:Zhiqi Cong, Osami Shoji, Chie Kasai, Norifumi Kawakami, Hiroshi Sugimoto, Yoshitsugu Shiro, and Yoshihito Watanabe

ACS Catalysis 2015 Volume 5(Issue 1) pp:150

Publication Date(Web):November 18, 2014

DOI:10.1021/cs501592f

The direct hydroxylation of alkanes under mild conditions is a key issue in catalytic chemistry that addresses an increasing number of industrial and economic requirements. Cytochrome P450s are monooxygenases that are capable of oxidizing less reactive C–H bonds; however, wild-type P450s are unavailable for many important nonnative substrates such as gaseous alkanes. Here, we report the enhanced hydroxylation activities and crystallographic evidence for the role of decoy molecules in wild-type P450BM3-catalyzed hydroxylation of gaseous ethane and propane by using the next generation of decoy molecule. A cocrystal structure of P450BM3 and a decoy molecule reveals that an N-perfluoroacyl amino acid (decoy molecule) partially occupies the substrate-binding site of P450BM3. This binding of the decoy re-forms the active site pocket to allow the accommodation of small substrates and simultaneously influences the formation of compound I species by expelling water molecules from the active site.Keywords: biocatalysis; cytochromes; decoy molecule; gaseous alkanes; hydroxylation

Acetate anion-triggered peroxygenation of non-native substrates by wild-type cytochrome P450s

Co-reporter:Hiroki Onoda, Osami Shoji and Yoshihito Watanabe

Dalton Transactions 2015 vol. 44(Issue 34) pp:15316-15323

Publication Date(Web):09 Jun 2015

DOI:10.1039/C5DT00797F

Cytochrome P450SPα (P450SPα) and cytochrome P450BSβ (P450BSβ) belonging to the CYP152 family of enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although P450SPα and P450BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as “decoy molecules.” Although we had assumed that acetic acid did not serve as a decoy molecule, P450SPα and P450BSβ efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450BSβ in the presence of 1 M acetate anion reached 590 ± 30 min−1.

Bringing out the Potential of Wild-type Cytochrome P450s using Decoy Molecules: Oxygenation of Nonnative Substrates by Bacterial Cytochrome P450s

Co-reporter:Osami Shoji;Yoshihito Watanabe

Israel Journal of Chemistry 2015 Volume 55( Issue 1) pp:32-39

Publication Date(Web):

DOI:10.1002/ijch.201400096

Abstract

To bring out the potential of wild-type cytochrome P450s, we have developed a series of “decoy molecules” to change their high substrate specificity without any mutagenesis. Decoy molecules are inert dummy substrates with structures that are very similar to those of natural substrates. The decoy molecules force long-alkyl-chain fatty acid hydroxylases (P450_BSβ, P450_SPα, and P450BM3) to generate the active species and to catalyze oxidation of various substrates other than fatty acids. Interestingly, the catalytic activity was highly dependent on the structure of decoy molecules. Furthermore, the enantioselectivity of reactions catalyzed by P450_BSβ and P450_SPα was also dependent on the structure of decoy molecules. The decoy molecule system allows us to control reactions catalyzed by wild-type enzymes by designing decoy molecules.

Innentitelbild: Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp) Bound to Synthetic Metal Complexes (Angew. Chem. 11/2014)

Co-reporter:Chikako Shirataki;Dr. Osami Shoji;Mitsuyoshi Terada;Dr. Shin-ichi Ozaki;Dr. Hiroshi Sugimoto;Dr. Yoshitsugu Shiro;Dr. Yoshihito Watanabe

Angewandte Chemie 2014 Volume 126( Issue 11) pp:

Publication Date(Web):

DOI:10.1002/ange.201401090

Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp) Bound to Synthetic Metal Complexes

Co-reporter:Chikako Shirataki;Dr. Osami Shoji;Mitsuyoshi Terada;Dr. Shin-ichi Ozaki;Dr. Hiroshi Sugimoto;Dr. Yoshitsugu Shiro;Dr. Yoshihito Watanabe

Angewandte Chemie International Edition 2014 Volume 53( Issue 11) pp:2862-2866

Publication Date(Web):

DOI:10.1002/anie.201307889

Abstract

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA_p) can capture several synthetic metal complexes other than heme. The crystal structures of HasA_p harboring synthetic metal complexes revealed only small perturbation of the overall HasA_p structure. An inhibitory effect upon heme acquisition by HasA_p bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA_p bound to iron–phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA_p as an iron source.

Inside Cover: Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp) Bound to Synthetic Metal Complexes (Angew. Chem. Int. Ed. 11/2014)

Co-reporter:Chikako Shirataki;Dr. Osami Shoji;Mitsuyoshi Terada;Dr. Shin-ichi Ozaki;Dr. Hiroshi Sugimoto;Dr. Yoshitsugu Shiro;Dr. Yoshihito Watanabe

Angewandte Chemie International Edition 2014 Volume 53( Issue 11) pp:

Publication Date(Web):

DOI:10.1002/anie.201401090

Inhibition of Heme Uptake in Pseudomonas aeruginosa by its Hemophore (HasAp) Bound to Synthetic Metal Complexes

Co-reporter:Chikako Shirataki;Dr. Osami Shoji;Mitsuyoshi Terada;Dr. Shin-ichi Ozaki;Dr. Hiroshi Sugimoto;Dr. Yoshitsugu Shiro;Dr. Yoshihito Watanabe

Angewandte Chemie 2014 Volume 126( Issue 11) pp:2906-2910

Publication Date(Web):

DOI:10.1002/ange.201307889

Abstract

The heme acquisition system A protein secreted by Pseudomonas aeruginosa (HasA_p) can capture several synthetic metal complexes other than heme. The crystal structures of HasA_p harboring synthetic metal complexes revealed only small perturbation of the overall HasA_p structure. An inhibitory effect upon heme acquisition by HasA_p bearing synthetic metal complexes was examined by monitoring the growth of Pseudomonas aeruginosa PAO1. HasA_p bound to iron–phthalocyanine inhibits heme acquisition in the presence of heme-bound HasA_p as an iron source.

Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules

Co-reporter:Norifumi Kawakami, Osami Shoji and Yoshihito Watanabe

Chemical Science 2013 vol. 4(Issue 6) pp:2344-2348

Publication Date(Web):15 Mar 2013

DOI:10.1039/C3SC50378J

Cytochrome P450BM3 (P450BM3) is a long-alkyl-chain fatty acid hydroxylase that shows an extremely high catalytic turnover rate and high coupling efficiency of NADPH for product formation (product formation rate per NADPH consumption rate). Although P450BM3 exclusively hydroxylates long-alkyl-chain fatty acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inert dummy substrates (decoy molecules) turns P450BM3 into a small alkane hydroxylase. For example, PF-bound P450BM3 oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small alkane hydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid hydroxylation. In this study, we examined the experimental conditions for small alkane hydroxylation in an effort to improve the coupling efficiency and to realize the hydroxylation of their primary carbons. To increase the concentration of gaseous substrates in the reaction mixture, we performed reactions under the high-pressure condition of 0.5 MPa small alkanes. Propane hydroxylation under high-pressure conditions significantly improved the coupling efficiency to 48%. Furthermore, 1-propanol, which has never been observed under lower-pressure conditions, was produced. It is noteworthy that a detectable amount of ethanol was observed in the ethane hydroxylation under the pressure condition of 0.5 MPa, whereas methane was not hydroxylated. These results indicate that by increasing the concentration of small alkanes, the “P450BM3–decoy molecule system” can catalyze hydroxylation reactions of the primary carbons in small alkanes.

Highly Selective Hydroxylation of Benzene to Phenol by Wild-type Cytochrome P450BM3 Assisted by Decoy Molecules

Co-reporter:Dr. Osami Shoji;Tatsuya Kunimatsu;Dr. Norifumi Kawakami;Dr. Yoshihito Watanabe

Angewandte Chemie 2013 Volume 125( Issue 26) pp:6738-6742

Publication Date(Web):

DOI:10.1002/ange.201300282

Highly Selective Hydroxylation of Benzene to Phenol by Wild-type Cytochrome P450BM3 Assisted by Decoy Molecules

Co-reporter:Dr. Osami Shoji;Tatsuya Kunimatsu;Dr. Norifumi Kawakami;Dr. Yoshihito Watanabe

Angewandte Chemie International Edition 2013 Volume 52( Issue 26) pp:6606-6610

Publication Date(Web):

DOI:10.1002/anie.201300282

Acetate anion-triggered peroxygenation of non-native substrates by wild-type cytochrome P450s

Co-reporter:Hiroki Onoda, Osami Shoji and Yoshihito Watanabe

Dalton Transactions 2015 - vol. 44(Issue 34) pp:NaN15323-15323

Publication Date(Web):2015/06/09

DOI:10.1039/C5DT00797F

Cytochrome P450SPα (P450SPα) and cytochrome P450BSβ (P450BSβ) belonging to the CYP152 family of enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although P450SPα and P450BSβ have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as “decoy molecules.” Although we had assumed that acetic acid did not serve as a decoy molecule, P450SPα and P450BSβ efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450BSβ in the presence of 1 M acetate anion reached 590 ± 30 min−1.

Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules

Co-reporter:Norifumi Kawakami, Osami Shoji and Yoshihito Watanabe

Chemical Science (2010-Present) 2013 - vol. 4(Issue 6) pp:NaN2348-2348

Publication Date(Web):2013/03/15

DOI:10.1039/C3SC50378J

Cytochrome P450BM3 (P450BM3) is a long-alkyl-chain fatty acid hydroxylase that shows an extremely high catalytic turnover rate and high coupling efficiency of NADPH for product formation (product formation rate per NADPH consumption rate). Although P450BM3 exclusively hydroxylates long-alkyl-chain fatty acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inert dummy substrates (decoy molecules) turns P450BM3 into a small alkane hydroxylase. For example, PF-bound P450BM3 oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small alkane hydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid hydroxylation. In this study, we examined the experimental conditions for small alkane hydroxylation in an effort to improve the coupling efficiency and to realize the hydroxylation of their primary carbons. To increase the concentration of gaseous substrates in the reaction mixture, we performed reactions under the high-pressure condition of 0.5 MPa small alkanes. Propane hydroxylation under high-pressure conditions significantly improved the coupling efficiency to 48%. Furthermore, 1-propanol, which has never been observed under lower-pressure conditions, was produced. It is noteworthy that a detectable amount of ethanol was observed in the ethane hydroxylation under the pressure condition of 0.5 MPa, whereas methane was not hydroxylated. These results indicate that by increasing the concentration of small alkanes, the “P450BM3–decoy molecule system” can catalyze hydroxylation reactions of the primary carbons in small alkanes.

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 produced by one-point mutagenesis and peroxygenation of non-native substrates

Co-reporter:Osami Shoji, Takashi Fujishiro, Kousuke Nishio, Yukiko Kano, Hiroshi Kimoto, Shih-Cheng Chien, Hiroki Onoda, Atsushi Muramatsu, Shota Tanaka, Ayumi Hori, Hiroshi Sugimoto, Yoshitsugu Shiro and Yoshihito Watanabe

Catalysis Science & Technology (2011-Present) 2016 - vol. 6(Issue 15) pp:NaN5811-5811

Publication Date(Web):2016/04/22

DOI:10.1039/C6CY00630B

A substrate-binding-state mimic of H2O2-dependent cytochrome P450 that is able to catalyze monooxygenation of non-native substrates was constructed by one-point mutagenesis of P450SPα (CYP152B1). P450SPα, a long-alkyl-chain fatty acid hydroxylase, lacks any general acid–base residue around the heme. The carboxylate group of a fatty acid is thus indispensable for the generation of active species using H2O2. We prepared an A245E mutant to mimic a substrate-binding state by placing a carboxylate group at the active site. The active site structure of the A245E mutant is similar to that of the fatty-acid-bound state of P450SPα and catalyzes styrene oxidation at a rate of 280 min−1 (kcat), whereas the wild-type enzyme does not show any catalytic activity. More importantly, the same mutation, i.e. the mutation of the highly conserved threonine in P450s to glutamic acid, was also effective in introducing peroxygenase activity into P450BM3, P450cam, and CYP119. These results indicate that a variety of peroxygenases based on P450s can be constructed by one-point mutagenesis.

Control of stereoselectivity of benzylic hydroxylation catalysed by wild-type cytochrome P450BM3 using decoy molecules

Co-reporter:Kazuto Suzuki, Joshua Kyle Stanfield, Osami Shoji, Sota Yanagisawa, Hiroshi Sugimoto, Yoshitsugu Shiro and Yoshihito Watanabe

Catalysis Science & Technology (2011-Present) 2017 - vol. 7(Issue 15) pp:NaN3338-3338

Publication Date(Web):2017/06/30

DOI:10.1039/C7CY01130J

The hydroxylation of non-native substrates catalysed by wild-type P450BM3 is reported, wherein “decoy molecules”, i.e., native substrate mimics, controlled the stereoselectivity of hydroxylation reactions. We employed decoy molecules with diverse structures, resulting in either a significant improvement in enantioselectivity or clear inversion of stereoselectivity in the benzylic hydroxylation of alkylbenzenes and cycloalkylbenzenes. For example, supplementation of wild-type P450BM3 with 5-cyclohexylvaleric acid-L-phenylalanine (5CHVA-Phe) and Z-proline-L-phenylalanine yielded 53% (R) ee and 56% (S) ee for indane hydroxylation, respectively, although 16% (S) ee was still observed in the absence of any additives. Moreover, we performed a successful crystal structure analysis of 5CHVA-L-tryptophan-bound P450BM3 at 2.00 Å, which suggests that the changes in selectivity observed were caused by conformational changes in the enzyme induced by binding of the decoy molecules.