Epoxidases

Like the a2ole derivatives, it inhibits the biosynthesis of ergosterol. However, naftifine [65472-88-0] does not inhibit the cytochrome P-450 dependent C-14-demethylase, but the epoxidation of squalene. Squalene epoxidase cataly2es the first step in the conversion of squalene via lanosterol to ergosterol in yeasts and fungi or to cholesterol in mammalian cells. The squalene epoxidase in C. albicans is 150 times more sensitive to naftifine, C2 H2 N, than the en2yme in rat fiver (15). Naftifine is available as a 1% cream. 

Lanosterol biosynthesis begins with the selective conversion of squalene to its epoxide, (35)-2,3-oxidosqualene/ catalyzed by squalene epoxidase. Molecular 02 provides the source of the epoxide oxygen atom, and NADPH is required, along with a flavin coenzyme. The proposed mechanism involves... 

Squalene epoxidase, a key enzyme in the biosynthesis of cholesterol (9), epoxidizes one face of one of the three different olefins in squalene (7) to give squalene epoxide (8), which then cyclizes eventually to give cholesterol (9) (Scheme 1). The AD of squalene (7)... 

Squalene epoxidase, like most enzymes responsible for the later steps of sterol biosynthesis [43, 51], is membrane-bound which makes its purification in native form challenging. The purification is additionally complicated by the presence of a large number of cytochrome P450 and other enzymes that have similar hydro-phobicity and size as squalene epoxidase and are hence difficult to remove [52]. Most studies have been carried out with rat liver microsome squalene epoxidase either partially purified or as a homogenate of the cell membrane fraction. In vitro reconstitution of squalene epoxidase activity is absolutely dependent on molecular oxygen, NADPH, FAD, and NADPH-cytochrome c reductase [52, 53]. In this respect, squalene epoxidase resembles the cytochrome P450 enzymes described... 

To summarize, squalene epoxidase is a flavoprotein capable of catalyzing the insertion of oxygen into the 2,3-double bond of squalene to give 2,3-oxidosqualene, with the second oxygen atom from 02 being reduced to water. The reducing equivalents necessary for this transformation are relayed from NADPH through NADPH-cytochrome c reductase to the flavin cofactor of the epoxidase. 

Like the examples above, dihydroxyacetanilide epoxidase (DHAE) uses an olefin as the substrate for epoxidation. Its mechanism, however, is fundamentally different from those of cytochrome P450 or flavin-dependent enzymes. Dihydroxyacetanilide is an intermediate in the biosynthesis of the epoxyquinones LL-C10037a, an antitumor agent produced by the actinomycete Streptomyces LL-C10037 [75, 76], and MM14201, an antibiotic produced by Streptomyces MPP 3051 (Scheme 10.20) [77]. The main structural difference between the two antibiotics lies in the opposite stereochemistry of the oxirane ring. 

Scheme 10.26 Partial biosynthetic pathway of fosfomycin and bialaphos. Both pathways use a homologous set of enzymes for the synthetic steps leading from phosphoenolpyruvate (PEP) to phosphonoacetaldehyde (PAA). The conversion of hydroxypropylphosphonic acid (HPP) to fosfomycin is catalyzed by the epoxidase HppE. Propenylphosphonic acid (PPA), however, is not converted to fosfomycin.

Vitamin K carboxylase is a transmembraneous protein in the lipid bilayer of the endoplasmatic reticulum (ER). It is highly glycosilated and its C-terminal is on the luminal side of the membrane. Besides its function as carboxylase it takes part as an epoxidase in the vitamin K cycle (Fig. 1). For the binding of the y-carboxylase the vitamin K-dependent proteins have highly conserved special recognition sites. Most vitamin K-dependent proteins are carboxy-lated in the liver and in osteoblasts, but also other tissues might be involved, e.g., muscles. 

Hager, A Perz, H. (1970). Veranderung der lichtabsorption eines carotenoids im enzym (de-epoxidase)-substrat (violaxanthin)-komplex. Planta, 93, 314-22. 

Terbinafme (Fig. 5.171), a member ofthe allylamine class ofantimycotics, is an inhibitor of the enzyme squalene epoxidase in fungal ergosterol biosynthesis. Terbinafme is orally active, is fungicidal and is effective against a broad range of dermatophytes and yeasts. It can also be used topically as a cream. 

The synthetic thiocarbamates, of which tolnaftate (Fig. 5.20J) is an example, also inhibit squalene epoxidase. Tolnaftate inhibits this enzyme from C. albicans, but is inactive against whole cells, presumably because of its inability to penetrate the cell wall. Tolnaftate is used topically in the treatment or prophylaxis of tinea. 

This synthetic allylamine derivative inhibits the enzyme squalene epoxidase at an early stage in fungal sterol biosynthesis. Acting as a structural analogue of squalene, naffidine causes the accumulation of this unsaturated hydrocarbon, and a decrease in ergosterol in the fungal cell membrane. 

The oxidation of carotenes results in the formation of a diverse array of xanthophylls (Fig. 13.7). Zeaxanthin is synthesised from P-carotene by the hydroxylation of C-3 and C-3 of the P-rings via the mono-hydroxylated intermediate P-cryptoxanthin, a process requiring molecular oxygen in a mixed-function oxidase reaction. The gene encoding P-carotene hydroxylase (crtZ) has been cloned from a number of non-photosynthetic prokaryotes (reviewed by Armstrong, 1994) and from Arabidopsis (Sun et al, 1996). Zeaxanthin is converted to violaxanthin by zeaxanthin epoxidase which epoxidises both P-rings of zeaxanthin at the 5,6 positions (Fig. 13.7). The... 

CRTR-e = e-ring hydroxyiase CRTR-b = p-ring hydroxylase ZEP-1 = zeaxanthin epoxidase NXS = neoxanthin synthase VDE 1 = violaxanthin de-epoxidase. 

BOUVIER F, D HARLINGUE A, HUGUENEY P, MARIN E, MARION-POLL A and CAMARA B (1996) Xanthophyll biosynthesis cloning, expression, functional reconstitution and regulation of 3-cyclohexenyl carotenoid epoxidase from pepper Capsicum annuum) , J Biol Chem, 271, 28861-7. 

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