Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium.

Narhi, Linda O.; Fulco, Armand J.

doi:10.1016/s0021-9258(17)38369-2

Cited by 575 publications

(175 citation statements)

References 29 publications

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“…P450-mediated enzymatic oxidation reactions can be used for the efficient generation of alcohols from alkanes, while these conversions are not feasible using current organic synthetic methods (57)(58)(59)(60). The catalytic efficiencies of class III P450 proteins, which are fused proteins of P450 and its reductase, are higher than those of other P450 classes (16)(17)(18)(19)(20)58). In the present study, CYP505D6 was shown to be versatile, generating fatty acids hydroxylated at more positions than those produced by CYP102A1 and CYP505A1.…”

Section: Discussionmentioning

confidence: 61%

“…In our previous study, the absorption spectra of native and recombinant CYP505A1 enzymes were determined. However, a broad shoulder in the 440-to 490-nm region derived from flavin prosthetic groups (16) was not prominent because of low flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) contents (17,41). The absorption spectra of recombinant CYP505D6 were similar to those of CYP505A1.…”

Section: Resultsmentioning

confidence: 99%

“…CYP505A1 catalyzes the subterminal (-1 to -3) hydroxylation of fatty acids (16)(17)(18), whereas Krac9955 hydroxylates tetradecanoic acid at the -1 to -7 positions (21). In CYP102A1, Tyr51 replaces Phe51 and Val51 in CYP505A1 and Krac9955, respectively.…”

Section: Discussionmentioning

confidence: 99%

“…One unit of activity was defined as the amount of enzyme that catalyzes the reduction of cytochrome c at an initial rate of 1 pmol min Ϫ1 . The rate of cytochrome c reduction was determined spectrophotometrically at 30°C using 21.1 mM Ϫ1 cm Ϫ1 as the extinction coefficient for reduced cytochrome c minus that for oxidized cytochrome c at 550 nm (16)(17)(18)(19)59).…”

Section: Methodsmentioning

confidence: 99%

“…Among them, seven homologous genes encode fused proteins of cytochrome P450 and its reductase (11). P450BM3 (CYP102A1) of Bacillus megaterium and P450foxy (CYP505A1) of Fusarium oxysporum are also fusions of cytochrome P450 and its reductase and catalyze subterminal (-1 to -3) hydroxylation of fatty acids (16)(17)(18). Not only fatty acids but also fatty alcohols and amides are hydroxylated by CYP102A1 (19,20).…”

mentioning

confidence: 99%

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Biochemical Characterization of CYP505D6, a Self-Sufficient Cytochrome P450 from the White-Rot Fungus Phanerochaete chrysosporium

Sakai

Matsuzaki

Wise

et al. 2018

Appl Environ Microbiol

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The activity of a self-sufficient cytochrome P450 enzyme CYP505D6 from the lignin-degrading basidiomycete was characterized. Recombinant CYP505D6 was produced in and purified. In the presence of NADPH, CYP505D6 used a series of saturated fatty alcohols with C9-18 carbon chain lengths as substrates. Hydroxylation occurred at ω-1 to ω-6 positions of such substrates with C9-15 carbon chain lengths, except for 1-dodecanol, which was hydroxylated at ω-1 to ω-7 positions. Fatty acids were also substrates of CYP505D6. Based on the sequence alignment, the corresponding amino acid of Tyr51, which is located at the entrance to the active site pocket in CYP102A1, was Val51 in CYP505D6. To understand the diverse hydroxylation mechanism, wild-type CYP505D6 and its V51Y variant, and wild-type CYP102A1 and its Y51V variant were generated, and the products of their reaction with dodecanoic acid analyzed. Compared with the wild-type CYP505D6, its V51Y variant generated fewer products hydroxylated at ω-4 to ω-6 positions. The products generated by the wild-type CYP102A1 were hydroxylated at ω-1 to ω-4 positions, whereas its Y51V variant generated ω-1 to ω-7 hydroxydodecanoic acids. These observations indicated that Val51 plays an important role in determining the regio-specificity of fatty acid hydroxylation, at least that at ω-4 to ω-6 positions. Aromatic compounds, such as naphthalene and 1-naphthol, were also hydroxylated by CYP505D6. These findings highlight a unique broad substrate spectrum of CYP505D6, rendering it an attractive candidate enzyme for the biotechnological industry. is a white-rot fungus whose metabolism of lignin, aromatic pollutants, and lipids has been most extensively studied. This fungus harbors 154 cytochrome P450-encoding genes in the genome. As evidenced in the current study, CYP505D6, a fused protein of P450 and its reductase, hydroxylates fatty alcohols (C9-15) and fatty acids (C9-15) at ω-1 to ω-7 or ω-6 positions, respectively. Naphthalene and 1-naphthol were also hydroxylated, indicating that the substrate specificity of CYP505D6 is broader than that of the known fused proteins CYP102A1 and CYP505A1. Substrate versatility of CYP505D6 makes this enzyme an attractive candidate for biotechnological applications.

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