Publications

• Aromatic C-H bond hydroxylation by P450 peroxygenases: a facile colorimetric assay for monooxygenation activities of enzymes based on Russig's blue formation.

  Shoji O., Wiese C., Fujishiro T., Shirataki C., Wunsch B., Watanabe Y.

  Aromatic C-H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450BSbeta (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation o ... >> More

  Aromatic C-H bond hydroxylation of 1-methoxynaphthalene was efficiently catalyzed by the substrate misrecognition system of the hydrogen peroxide dependent cytochrome P450BSbeta (CYP152A1), which usually catalyzes hydroxylation of long-alkyl-chain fatty acids. Very importantly, the hydroxylation of 1-methoxynaphthalene can be monitored by a color change since the formation of 4-methoxy-1-naphthol was immediately followed by its further oxidation to yield Russig's blue. Russig's blue formation allows us to estimate the peroxygenation activity of enzymes without the use of high performance liquid chromatography, gas chromatography, and nuclear magnetic resonance measurements. << Less

  J Biol Inorg Chem 15:1109-1115(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Direct involvement of hydrogen peroxide in bacterial alpha-hydroxylation of fatty acid.

  Matsunaga I., Yamada M., Kusunose E., Nishiuchi Y., Yano I., Ichihara K.

  We have reported that fatty-acid alpha-hydroxylase partially purified from Sphingomonas paucimobilis required NADH and molecular oxygen. In this study, we found that the reaction was greatly inhibited by catalase. Glutathione and glutathione peroxidase also inhibited alpha-hydroxylation, but super ... >> More

  We have reported that fatty-acid alpha-hydroxylase partially purified from Sphingomonas paucimobilis required NADH and molecular oxygen. In this study, we found that the reaction was greatly inhibited by catalase. Glutathione and glutathione peroxidase also inhibited alpha-hydroxylation, but superoxide dismutase and mannitol did not. Replacement of NADH and molecular oxygen by hydrogen peroxide increased the alpha-hydroxylation activity. In the presence of hydrogen peroxide, molecular oxygen was not required for the activity. These findings suggest that hydrogen peroxide was essential for bacterial alpha-hydroxylase. << Less

  FEBS Lett 386:252-254(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Peroxide-utilizing biocatalysts: structural and functional diversity of heme-containing enzymes.

  Matsunaga I., Shiro Y.

  Heme-containing enzymes, such as peroxidases, catalase and peroxygenase P450 all utilize peroxides for their specific reactions. A variety of reactions catalyzed by such heme-containing enzymes involve a common, highly reactive intermediate, the so-called compound I (oxo-ferryl porphyrin pi-cation ... >> More

  Heme-containing enzymes, such as peroxidases, catalase and peroxygenase P450 all utilize peroxides for their specific reactions. A variety of reactions catalyzed by such heme-containing enzymes involve a common, highly reactive intermediate, the so-called compound I (oxo-ferryl porphyrin pi-cation radical), which is generated via the reaction of peroxide with a ferric heme iron. However, the main reaction catalyzed by the heme-containing enzyme is determined by the accessibility of substrates to their active sites. Using the accumulated knowledge, we delineate a view, in which machineries of the heme-containing enzymes, especially the heme distal side structures, precisely regulate their functions in terms of sharing a common reactive intermediate. We also show the possibility that a hemoprotein of one functionality can be engineered to that with another functionality by modifying the heme distal side elements, on the basis of molecular-based mechanistic and structural data on these peroxide-utilizing enzymes. << Less

  Curr Opin Chem Biol 8:127-132(2004) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Unique heme environment at the putative distal region of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis (peroxygenase P450(SPalpha).

  Imai Y., Matsunaga I., Kusunose E., Ichihara K.

  Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P45 ... >> More

  Fatty acid alpha-hydroxylase from Sphingomonas paucimobilis is a hydrogen peroxide-dependent cytochrome P450 (P450) enzyme (P450(SPalpha)). In this study, heme-ligand exchange reactions of P450(SPalpha) were investigated using the optical spectroscopic method and compared with those of various P450s. Alkylamines (C >/= 5) induced changes in the spectrum of ferric P450(SPalpha) to one typical of a nitrogenous ligand-bound low-spin form of ferric P450, although their affinities were lower than those for other P450s, and a substrate, laurate, did not interfere with the binding in contrast with in the cases of other P450s. Other compounds having a nitrogen donor atom to the heme iron of P450, including pyridine or 1-methylimidazole, induced no change in the spectrum of P450(SPalpha) in either the ferric or ferrous state. Practically no spectral change was observed on the addition of alkyl isocyanides to ferric P450s. On the other hand, cyanide induced a change in the spectrum of ferric P450(SPalpha) to one characteristic of cyanide-bound form of ferric P450. The affinity of cyanide increased when the substrate was added, in contrast with in the cases of other P450s. Ferrous P450(SPalpha) combined with CO and alkyl isocyanides, and the affinity for CO was of the same order of magnitude as in the cases of other P450s. These findings suggest a unique heme environment of P450(SPalpha), in which most compounds usually acting as external ligands of ferric P450s are prevented from gaining access to the heme iron of P450(SPalpha). The unique properties of the hydroxylase reaction catalyzed by P450(SPalpha), where an oxygen atom of hydrogen peroxide but not of molecular oxygen is utilized and incorporated into a fatty acid at its alpha position, is possibly related with such a specific heme environment of this P450. A possible mechanism for the peroxygenase reaction of P450(SPalpha) is proposed. << Less

  J Biochem 128:189-194(2000) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Substrate recognition and molecular mechanism of fatty acid hydroxylation by cytochrome P450 from Bacillus subtilis: crystallographic, spectroscopic and mutational studies.

  Lee D.-S., Yamada A., Sugimoto H., Matsunaga I., Ogura H., Ichihara K., Adachi S., Park S.-Y., Shiro Y.

  Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450(BSbeta) ... >> More

  Cytochrome P450 isolated from Bacillus subtilis (P450(BSbeta); molecular mass, 48 kDa) catalyzes the hydroxylation of a long-chain fatty acid (e.g. myristic acid) at the alpha- and beta-positions using hydrogen peroxide as an oxidant. We report here on the crystal structure of ferric P450(BSbeta) in the substrate-bound form, determined at a resolution of 2.1 A. P450(BSbeta) exhibits a typical P450 fold. The substrate binds to a specific channel in the enzyme and is stabilized through hydrophobic interactions of its alkyl side chain with some hydrophobic residues on the enzyme as well as by electrostatic interaction of its terminal carboxylate with the Arg(242) guanidium group. These interactions are responsible for the site specificity of the hydroxylation site in which the alpha- and beta-positions of the fatty acid come into close proximity to the heme iron sixth site. The fatty acid carboxylate group interacts with Arg(242) in the same fashion as has been reported for the active site of chloroperoxidase, His(105)-Glu(183), which is an acid-base catalyst in the peroxidation reactions. On the basis of these observations, a possible mechanism for the hydroxylation reaction catalyzed by P450(BSbeta) is proposed in which the carboxylate of the bound-substrate fatty acid assists in the cleavage of the peroxide O-O bond. << Less

  J. Biol. Chem. 278:9761-9767(2003) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Further characterization of hydrogen peroxide-dependent fatty acid alpha-hydroxylase from Sphingomonas paucimobilis.

  Matsunaga I., Yamada M., Kusunose E., Miki T., Ichihara K.

  Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis ... >> More

  Although fatty acid alpha-hydroxylase (FAAH) activity has been detected in various species, FAAH has not been sufficiently characterized. In this report, we describe the properties of FAAH highly purified from Sphingomonas paucimobilis. The FAAH was purified by about 5,200-fold. Blotting analysis with a specific antibody against the FAAH showed that its apparent molecular mass was approximately 43 kDa. FAAH showed alpha-hydroxylation activity in the presence of H2O2, but little if any activity with cumene hydroperoxide, t-butyl hydroperoxide, or t-butyl peroxybenzonate. The Km value for H2O2 was 72 microM. Highly purified FAAH oxidized various non-esterified saturated and unsaturated fatty acids including myristic acid, but not myristoyl-CoA. Potassium cyanide and sodium azide inhibited the FAAH activity in a concentration-dependent manner. Other respiratory chain inhibitors such as rotenone and antimycin A did not inhibit the activity. Among cytochrome P450 inhibitors, SKF-525A markedly inhibited the activity at the concentration of 2 mM, but CO did not. Imidazole, an inhibitor of plant alpha-oxidation, showed no inhibitory effect at 1 mM. << Less

  J Biochem 124:105-110(1998) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Enzymatic reaction of hydrogen peroxide-dependent peroxygenase cytochrome P450s: kinetic deuterium isotope effects and analyses by resonance Raman spectroscopy.

  Matsunaga I., Yamada A., Lee D.S., Obayashi E., Fujiwara N., Kobayashi K., Ogura H., Shiro Y.

  Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acid ... >> More

  Cytochromes P450SP(alpha) (CYP152B1) and P450BS(beta) (CYP152A1), which are isolated from Sphingomonas paucimobilis and Bacillus subtilis, respectively, belong to the P450 superfamily, but catalyze hydroxylation reactions, in which an oxygen atom from H2O2 is efficiently introduced into fatty acids (e.g., myristic acid). P450SP(alpha) produces the alpha-hydroxylated (alpha-OH) products at 100%, while P450BS(beta) produces alpha- and beta-hydroxylated (beta-OH) products at 33 and 67%, respectively. Using deuterium-substituted fatty acids ([2,2-d2]-myristic acid and d27-myristic acid) as a substrate, the peroxygenase reactions of the two bacterial P450s were investigated. In the P450SP(alpha) reaction, we observed an intermolecular noncompetitive kinetic isotope effect on Vmax (DV = 4.1) when [2,2-d2]-myristic acid was used, suggesting that an isotopically sensitive step involving the alpha-hydrogen of the fatty acid is present in the catalytic cycle. On the other hand, D(V/K) was masked, in sharp contrast to the features of usual monooxygenases P450. The characteristic kinetic features can be interpreted in terms of the faster product formation than the substrate dissociation. A similar kinetic isotope effect was observed [DV = 4.9, D(V/K) approximately 1] for the P450BS(beta) reaction, when d27-myristic acid was used as a substrate, indicating that the reaction mechanism is the same for both peroxygenases. The resonance Raman spectral data of P450BS(beta) in the ferric and ferrous-CO forms in the presence and absence of myristic acid demonstrated that the catalytic pocket of the enzyme is polar, so that the location of the carboxylate of the substrate close to the sixth ligand of the heme could be allowed. On the basis of these results on the kinetic isotope effects and spectroscopy, we discuss the possible mechanisms of the alpha- and beta-hydroxylation of fatty acids catalyzed by peroxygenases P450SP(alpha) and P450BS(beta). << Less

  Biochemistry 41:1886-1892(2002) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Characterization of the ybdT gene product of Bacillus subtilis: novel fatty acid beta-hydroxylating cytochrome P450.

  Matsunaga I., Ueda A., Fujiwara N., Sumimoto T., Ichihara K.

  We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa ... >> More

  We have characterized the gene encoding fatty acid alpha-hydroxylase, a cytochrome P450 (P450) enzyme, from Sphingomonas paucimobilis. A database homology search indicated that the deduced amino acid sequence of this gene product was 44% identical to that of the ybdT gene product that is a 48 kDa protein of unknown function from Bacillus subtilis. In this study, we cloned the ybdT gene and characterized this gene product using a recombinant enzyme to clarify function of the ybdT gene product. The carbon monoxide difference spectrum of the recombinant enzyme showed the characteristic one of P450. In the presence of H2O2, the recombinant ybdT gene product hydroxylated myristic acid to produce beta-hydroxymyristic acid and alpha-hydroxymyristic acid which were determined by high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry. The amount of these products increased with increasing reaction period and amount of H2O2 in the reaction mixture. The amount of beta-hydroxyl product was slightly higher than that of alpha-hydroxyl product at all times during the reaction. However, no reaction products were detected at any time or at any concentration of H2O2 when heat-inactivated enzyme was used. HPLC analysis with a chiral column showed that the beta-hydroxyl product was nearly enantiomerically pure R-form. These results suggest that this P450 enzyme is involved in a novel biosynthesis of beta-hydroxy fatty acid. << Less

  Lipids 34:841-846(1999) [PubMed] [EuropePMC]

  This publication is cited by 4 other entries.