Bioconversion of substituted naphthalenes and β-eudesmol with the cytochrome P450 BM3 variant F87V

Abstract

Bioconversion of various substituted naphthalenes that contain 1-methoxy- and 1-ethoxy-naphthalenes, methylnaphthalenes, dimethylnaphthalenes, and naphthalenecarboxylic acid methyl esters were performed using recombinant Escherichia coli cells, which expressed the gene coding for a cytochrome P450 BM3 variant F87V (P450 BM3 (F87V)) that was N-terminally fused to an archaeal peptidyl-prolyl cis-trans isomerase. In addition, bioconversion experiments with the same substrates were carried out using those that expressed the phnA1A2A3A4 genes for a polycyclic aromatic hydrocarbon (PAH)-dihydroxylating dioxygenase, which originated from a PAH-utilizing marine bacterium Cycloclasticus sp. strain A5. Consequently, a variety of mono-hydroxylated derivatives were generated from these substituted naphthalenes. Oxidative aryl coupling was found to produce a novel compound 4,4'-diethoxy-[2,2']-binaphthalenyl-1,1'-diol from 1-ethoxynaphthalene with the E. coli cells expressing the P450 BM3 (F87V) gene. This recombinant E. coli was further shown to introduce the hydroxyl group regio- and stereo-specifically into a sesquiterpene β-eudesmol.

Role of substituents on the reactivity and product selectivity in reactions of naphthalene derivatives catalyzed by the orphan thermostable cytochrome P450, CYP175A1

“…Both of these two residues (e.g., Phe87 in cytochrome P450BM-3, and Leu80 in CYP175A1) extend into the heme pocket, and are positioned above the heme plane. Mutation of the Phe87 residue in cytochrome P450BM-3 showed alteration of the reaction specificity as well as of the enzymatic activity suggesting important role of this residue in the substrate association to the enzyme [17,[50][51][52][53][54]. Therefore, we mutated Leu80 residue in the CYP175A1 to phenylalanine residue (see Fig.…”

“…In order to further assess the role of the substrate binding pocket of the enzyme in the reactions of the substituted naphthalenes with H 2 O 2 , we compared the active site properties of CYP175A1 with that of CYP102A1 that is known to efficiently catalyze such reactions [17,18]. The crystal structure analyses (see Fig.…”

“…The crystal structure analyses showed that CYP102A1 (PDB code: 1FAG), CYP152A1 (PDB code: 1IZO) as well as CYP175A1 (PDB code: 1N97) consist of large substrate binding pockets located above the heme prosthetic group of the enzyme, suggesting that these enzymes would bind large substrates and show wide variations in the nature of the substrate [9]. CYP102A1 as well as CYP152A1 were reported to catalyze reactions of naphthalenes and analogs [17][18][19] supporting that the substrate binding cavity in CYP175A1 can indeed bind the aromatic hydrocarbon.…”

Production of Hydroxlated Flavonoids with Cytochrome P450 BM3 Variant F87V and Their Antioxidative Activities

“…A variant (mutant) of P450 BM3 with a Phe87Val (F87V) substitution [P450 BM3 (F87V)] has been shown to have promiscuous substrate specificity (affinity) for various small molecules, including aromatic compounds and sesquiterpenes, in the preparation of hydrogenated product(s) in previous studies. [1][2][3][4][5][6] The fusion protein, P450 BM3 (F87V) N-terminally fused to the PPIase, was much more soluble in E. coli cells when compared with the intact P450 BM3 (F87V) protein. 1) To the best of our knowledge, this P450 has not previously been shown to biotransform flavonoids, one of the two major families of plant pigments, flavonoids and carotenoids.…”

“…[1][2][3][4][5][6] The fusion protein, P450 BM3 (F87V) N-terminally fused to the PPIase, was much more soluble in E. coli cells when compared with the intact P450 BM3 (F87V) protein. 1) To the best of our knowledge, this P450 has not previously been shown to biotransform flavonoids, one of the two major families of plant pigments, flavonoids and carotenoids. Flavonoids have recently attracted considerable attention due to their beneficial effects on health, e.g., their antioxidative activity, 7) antimicrobial activity, 8) and estrogenic activity, 9) and are considered to be of medicinal and nutritional importance.…”