Cytochrome P450BM-3 (CYP102): Regiospecificity of oxidation of .omega.-unsaturated fatty acids and mechanism-based inactivation

Abstract

Cytochrome P450BM-3 preferentially oxidized fatty acids with terminal double or triple bonds to the omega-2 hydroxylated fatty acids rather than, respectively, to the epoxide or diacid metabolites. The enzyme is inactivated during catalytic turnover of long, terminally unsaturated fatty acids but not by the analogous medium-length fatty acids. Enzyme inactivation by 17-octadecynoic acid and 16-hydroxy-17-octadecynoic acid is due to alkylation of the prosthetic heme group to given an adduct tentatively identified as N-(2-oxo-3-hydroxy-17-carboxyheptadecyl)protoporphyrin IX by its chromatographic and spectroscopic properties. Catalytic turnover of 17-octadecenoic acid also results in heme modification. Fatty diacid monoethyl thioesters are introduced as a new class of irreversible inhibitors that exploit the omega-2 oxidation specificity of cytochrome P450BM-3. Catalytic oxidation of the monoethyl thioesters of dodecanedioic and hexadecanedioic acids results in enzyme inactivation and formation of the parent diacids as metabolites. Limited tryptic digestion of the enzyme after incubation with the monoethyl thioester of [14C]hexadecanedioic acid shows that the inactivating agent binds covalently to both the heme and flavin domains. This finding, and the observation that glutathione prevents inactivation of the enzyme by the monoethyl thioesters, indicate that a diffusible metabolite, probably the sulfoxide, is responsible for enzyme inactivation. The strong preference for omega-2 allylic or propargylic hydroxylation over terminal pi-bond oxidation is opposite to the usual cytochrome P450 pattern and requires that the enzyme actively suppress terminal pi-bond oxidation. The inference that the enzyme binds and sequesters the terminal carbon in a lipophilic pocket is consistent with the crystal structure of the hemoprotein domain of cytochrome P450BM-3.

Cytochrome P450 Epoxygenase Metabolism of Arachidonic Acid Inhibits Apoptosis

“…5B), similar to its effect in the wild-type cells. In BM3 cells pretreated with the P450 inhibitor 17-octadecynoic acid, previously shown to inhibit BM3 epoxygenase activity (18,51), administration of arachidonic acid increased ceramide levels (Fig. 5C) and induced apoptosis (Fig.…”

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

“…For unsaturated fatty acids from C 12 to C 22 , usually both, in-chain hydroxylation and epoxidation at the C=C-bond closest to the terminus were found (Fig. 3) [101][102][103]. The high activity for unsaturated fatty acids and branched fatty acids led to speculations whether these compounds constitute its natural (Tables 6, 7) [104,105].…”

“…The high activity for unsaturated fatty acids and branched fatty acids led to speculations whether these compounds constitute its natural (Tables 6, 7) [104,105]. Interestingly, whereas ω-C=C bonds increased the regioselectivity towards ω-2, probably due to the activated allylic location of hydroxylation (Table 6, entries 2, 3, and 9), a terminal alkyne-group was demonstrated to deactivate the enzyme by forming an adduct with the heme (Table 6, entry 10) [102,106]. The self-sufficiency of BM3 and its various reactions inspired the characterization of numerous additional CYP102 members, such as CYP102D1 from Streptomyces avermitiliswhich is the only characterized CYP102 outside the Bacillus family [107], CYP102A7 from Bacillus licheniformis (Table 5, entries 11, 30, and 40, Table 6, entries 8 and 13 and Table 7, entries 5 and 10) [78], and CYP102A2 and CYP102A3, both from Bacillus subtilis [108].…”

Productive Asymmetric Styrene Epoxidation Based on a Next Generation Electroenzymatic Methodology

“…As a result, space-time yields of cofactor mass transfer non-optimized systems are usually rather low with respect to the maximal activity of the production enzyme [16] because of fast competing side-reactions. For example, cytochrome P450cam coupled to the reductive electrochemical regeneration of putidaredoxin exhibited around 3% of the native activity with respect to 5-exo-hydroxy camphor formation from camphor, [30] cytochrom P450 BM3 regenerated by 1,10-dicarboxycobaltocene was able to hydroxylate lauric acid up to 3.6% of the theoretical rate, [29,31] and StyA coupled to the reductive electrochemical regeneration of FADH 2 exhibited only 0.6% of the native (S)-epoxidation activity towards styrene. [14] In comparison to the previously described electroenzymatic batch reaction system employing StyA, [14] we were able to exploit up to 8.7% of the native initial enzyme activity (2.1 U mg À1 ) [19] with respect to (S)-styrene oxide space-time yields, and up to 11.5% regarding overall space-time yields (Synthesis 2).…”