Sterol 14, demethylation reaction

Figure 5. Outline of the Sterol 14 Demethylation Reaction Sequence (9).

Fig. 6. 25 The major CYP51 substrates lanosterol, 24,25-dihydrolanosterol, and obtusifoliol. The upper panel shows the structures of CYP51 substrates lanosterol and 24,25-di-hydrolanosterol (substrates for mammalian and yeast CYPSls), and of the plant (and trypanosome) sterol substrate obtusifoliol [21, 447], The central panel shows the CYP51 sterol demethylation reaction that occurs by three consecutive oxidative reactions on the sterol 14a-methyl group to generate alcohol, then aldehyde, and then demethylation with release of formate. The lanosterol and 24,25-dihydrolanosterol demethylation products ultimately go on to form ergosterol and/or cholesterol. The obtusifoliol-demethylated product can ultimately be converted to sitosterol, or to other sterols including ergosterol. Cholesterol has an important role in Mtb infection and can be oxidized by Mtb P450s [356-358, 851], while ergosterol is the major membrane sterol in yeast [446, 852]...

Imazalil resistance appeared to be a stable character of the Isolates—no change in resistance level was observed after six weekly transfers on imazalil-free culture medium or six disease cycles in untreated lemon fruits. Six imazalil-resistant Isolates tested were cross-resistant to propiconazole, fenarimol, flusila-zole, penconazole, myclobutanil, prochloraz, diclobutrazol, and bltertanol, all of which inhibit the C-14 demethylation reaction in fungal sterol biosynthesis (27,28). However, the imazalil-resistant Isolates were sensitive to tridemorph and fenpropimorph, also inhibitors of sterol biosynthesis but of the A -A7 isomerization reaction (29). Imazalil-resistant mutants of P. italicum have shown the same pattern of cross-resistance (20,30). 

It was originally postulated that the methyl groups at C-4 were removed as COj -a suggestion that has proved to be correct. These groups are hydroxylated by a mixed-function oxidase which is NAD(P)H and Oj dependent. First, the 4a-methyl is attacked, yielding the 4 -hydroxymethyl-4 -methyl sterol. This reaction is catalyzed by a methyl sterol oxidase which has been solubilized and partially purified in Gaylor s laboratory [108]. The same enzyme preparation will, with reduced pyridine nucleotide and dioxygen, oxidize the C-30 carbon to a carboxylic acid. The 4a-methyl-4/8-hydroxymethyl-5a-cholestan-3j8-ol is not a substrate for sterol biosynthesis while its epimer is [5]. The detailed mechanisms for the enzymatic removal of C-30 and C-31 are not fully understood. The initial reaction yields a 4a-hydroxy-methyl sterol by inference however, neither the isolation nor the enzymatic formation of a 4a-hydroxymethyl sterol has been demonstrated in animal tissues. This may well result from the fact that the hydroxylation reaction is the slow step in the demethylation process [5]. 

Shyadehi AZ, DC Lamb, SL Kelly, DE Kelly, W-H Schunck, JN Wright, D Corina, M Akhtar (1996) The mechanism of the acyl-carbon bond cleavage reaction catalyzed by recombinant sterol 14a-demethyl-ase of Candida albicans (other names are lanosterol 14a-demethylase, P-450]4p, and CYP51). J Biol Chem 271 12445-12450. 

The last sequence of reactions in the biosynthesis of choles-terol involves approximately 20 enzymatic steps, starting with lanosterol. In mammals the major route involves a series of double-bond reductions and demethylations (fig. 20.10). The sequence of reactions involves reduction of the A24 double bond, the oxidation and removal of the 14a methyl group followed by the oxidation and removal of the two methyl groups at position 4 in the sterol. The final reaction is a reduction of the A7 double bond in 7-dehydro-cholesterol. An alternative pathway from lanosterol to cholesterol also exists. The enzymes involved in the transformation of lanosterol to cholesterol are all located on the endoplasmic reticulum. 

In summary, prochloraz, like other azoles, is an ergosterol biosynthesis inhibitor (i.e. it is an EB1) and it does this by blocking 14-demethylation (such compounds are sometimes referred to as demethylation inhibitors, or DMIs). As is now well known, these molecules bind to the sterol binding site of the demethylase enzyme in such a way as to allow the azole to bind to an iron atom in the active site (see below). The normal physiological reaction is therefore prevented. 

A complex series of reactions including opening of the cyloartenol cyclopropane ring, double bond formation and isomerization, demethylation of ring carbons, and methylation of the side chain result in formation of a number of different plant sterols [24]. Sitosterol is the most common plant sterol (Fig. 7) however, plants normally contain mixtures of sterols whose proportions differ from tissue to tissue. In addition, sterol esters, sterol... 

Lamb, D.C., D.E. Kelly, N.J. Manning, M.A. Kaderbhai, and S.L. Kelly (1999). Biodiversity of the P450 catalytic cycle Yeast cytochrome bj/ NADH cytochrome bj reductase complex efficiently drives the entire sterol 14-demethylation (CYP51) reaction. FEBS Lett. 462, 283-288. 

The 2,3-oxidosqualene cyclization reaction is one of particular Interest as a fungicide target since it is quite complicated and involves more than one TS or HE intermediate. It is surprising that more Inhibitors of the enzyme catalyzing this reaction have not been synthesized. A point of interest is the unusual overlap of specificity of 1-dodecyclimidazole (Figure 2) a an inhibitor of sterol C-14 demethylation and 2,3-oxidosqualene cyclization (12.323. The simple aliphatic substituent of this Imidazole may allow the compound to act as a TS analogue of 2,3-oxldosqualene and also to bind to the sterol C-14 demethylase and interact with the heme iron. A similar overlap of specificity... 

Two molecules of famesyl pyrophosphate are joined head-to-head to form squalene, a triterpene, in the first dedicated step towards sterol biosynthesis (Fig. 8.4). Squalene is then converted to 2,3-oxidosqualene, which next can be cyclized to the 30 carbon, 4-ring structure cycloartenol by the enzyme cycloartenol synthase (EC 5.4.99.8). Cycloartenol can be further modified by reactions such as desaturation or demethylation to form the common sterol backbones such as... 

The cytochrome P450-catalyzed cleavage of a carbon-carbon (C-C) bond has long been of interest because of the key role this transformation plays in the biosynthesis of cholesterol and all the sterol hormones derived from it. These reactions include the 14a-demethylation of lanosterol by CYP51, truncation of the cholesterol side chain... 

IDENTIFICATION OF THE COMPONENT REACTIONS OF OXIDATIVE STEROL C4-DEMETHYLATION... 

Two distinct component reactions of oxidative plant sterol C4-demethylation have been characterized. The 4a-carboxylic acid decarboxylase component will be further investigated. 

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