J. Neville Wright

The Mechanism of the Acyl-Carbon Bond Cleavage Reaction Catalyzed by Recombinant Sterol 14α-Demethylase of Candida albicans (Other Names Are: Lanosterol 14α-Demethylase, P-45014DM, and CYP51)

The Candida albicans sterol 14α-demethylase gene (P-45014DM, CYP51) was transferred to the yeast plasmid YEp51 placing it under the control of the GAL10 promoter. The resulting construct (YEp51:CYP51) when transformed into the yeast strain GRF18 gave a clone producing 1.5 μmol of P-450/liter of culture, the microsomal fraction of which contained up to 2.5 nmol of P-450/mg of protein. Two oxygenated precursors for the 14α-demethylase, 3β-hydroxylanost-7-en-32-al and 3β-hydroxylanost-7-en-32-ol, variously labeled with 2H and 18O at C-32 were synthesized. In this study the conversion of [32-2H,32-16O]- and [32-2H,32-18O]3β-hydroxylanost-7-en-32-al with the recombinant 14α-demethylase was performed under 16O2 or 18O2 and the released formic acid analyzed by mass spectrometry. The results showed that in the acyl-carbon bond cleavage step (i.e. the deformylation process) the original carbonyl oxygen at C-32 of the precursor is retained in formic acid and the second oxygen of formate is derived from molecular oxygen; precisely the same scenario that has previously been observed for the acyl-carbon cleavage steps catalyzed by aromatase (P-450arom) and 17α-hydroxylase-17,20-lyase (P-450,CYP17). In the light of these results the mechanism of the acyl-carbon bond cleavage step catalyzed by the 14α-demethylase is considered.

Incorporation of label from 18O2 into acetate during side-chain cleavage catalysed by cytochrome P-45017α(17α-hydroxylase-17,20-lyase)

Samples of pregnenolone and 17α-hydroxypregnenolone deuteriated at the C-21 methyl group have been prepared and subjected to side-chain cleavage with a pig testes microsomal preparation under 18O2. 17α-Hydroxypregnenolone was exclusively cleaved to dehydroisoandrosterone and the acetic acid released during the process was found to incorporate 0.90 atom of 18O. When a similar incubation was performed with pregnenolone two steroidal products, dehydroisoandrosterone and androsta-5,16-dien-3β-ol, were formed in an approximate ratio of 1:2–3 and the acetic acid formed in the process was again shown to contain 0.85–0.90 atom of 18O. Complementary experiments in which the two substrates labelled with 18O at the C-20 carbonyl group were incubated under 16O2 gave acetic acid retaining between 65–85% of the original carbonyl oxygen. The experiments strengthen the hypothesis that the two C(17)–C(20) bond cleavages described above correspond to the acyl-carbon fission process of the equation below showing the indicated fate of the various oxygen atoms: [graphic omitted]

Mechanistic consideration of P-450 dependent enzymic reactions: studies on oestriol biosynthesis

Methods for the synthesis of 19-hydroxy and 19-oxo derivatives of 16α-hydroxytestosterone [(11 b) and (12b) respectively] have been developed. These compounds were labelled with 18O, 2H, and 3H at C-19 and also with 3H at C-17. The conversion of [17α-3H]-16α,19-dihydroxytestosterone (11c) into oestriol (4c), using human placental aromatase was demonstrated in good yield and it was shown that in this process the 19-oxo compound (3c) is involved as an intermediate. The use of 16α,19-hydroxytestosterone, labelled with 3H predominantly in the HSi position, led to the conclusion that in oestriol biosynthesis the step, –CH2OH→–CHO, is accompanied by the loss of HRe and in the overall process the C-19 is ejected as HCOOH. On conducting experiments with either 18O2 or substrate containing 18O at C-19 it was shown that, in the conversion of 16α-hydroxy-19-oxotestosterone into oestriol, an atom of oxygen from O2 is incorporated into the formate. These features are similar to those already established for the corresponding biosynthesis of oestrone/oestradiol from androstenedione/testosterone. Our previous postulate that in oestrogen biosynthesis the same enzyme is involved in the hydroxylation reaction, –CH3→–CH2OH, and in the conversion, –CH2OH→–CHO, as well as in the final cleavage of the C-10–C-19 bond is further developed. Attention is drawn to the fact that, if cytochrome P-450 dependent reactions are viewed to occur via a radical mechanism, then a concept can be developed which unifies the wide variety of transformations catalysed by this group of enzymes. The diversity of reactions would then arise from the alternative mode of decomposition of radical species by one or a combination of the following processes: (a) hydrogen abstraction; (b) disproportionation; (c) fragmentation; and (d) association of radicals.

The cationic charges on Arg347, Arg358 and Arg449 of human cytochrome P450c17 (CYP17) are essential for the enzyme's cytochrome b5-dependent acyl-carbon cleavage activities.

CYP17 (17alpha-hydroxylase-17,20-lyase; also P450c17 or P450(17alpha)) catalyses the17alpha-hydroxylation of progestogens and the subsequent acyl-carbon cleavage of the 17alpha-hydroxylated products (lyase activity) in the biosynthesis of androgens. The enzyme also catalyses another type of acyl-carbon cleavage (direct cleavage activity) in which the 17alpha-hydroxylation reaction is by-passed. Human CYP17 is heavily dependent on the presence of the membrane form of cytochrome b(5) for both its lyase and direct cleavage activities. In the present study it was found that substitution of human CYP17 amino acids, Arg(347), Arg(358) and Arg(449), with non-cationic residues, yielded variants that were impaired in the two acyl-carbon bond cleavage activities, quantitatively to the same extent and these were reduced to between 3 and 4% of the wild-type protein. When the arginines were replaced by lysines, the sensitivity to cytochrome b(5) was restored and the acyl-carbon cleavage activities were recovered. All of the human mutant CYP17 proteins displayed wild-type hydroxylase activity, in the absence of cytochrome b(5). The results suggest that the bifurcated cationic charges at Arg(347), Arg(358) and Arg(449) make important contributions to the formation of catalytically competent CYP17.cytochrome b(5) complex. The results support our original proposal that the main role of cytochrome b(5) is to promote protein conformational changes which allow the iron-peroxo anion to form a tetrahedral adduct that fragments to produce the acyl-carbon cleavage products.

Mechanistic studies on pregnene side-chain cleavage enzyme (17α-hydroxylase-17,20-lyase) using 18O

In the conversion of pregnenolone 1 into dehydroisoandrosterone 3 and 3β-hydroxyandrosta-5,16-diene 5 an atom of oxygen from O2 is incorporated into the side chain released as acetate; during the conversions the C-21 methyl hydrogens of the precursor are not disturbed, the status of 16α and 17α hydrogen atoms during the conversions is also defined.

The mechanism of cytochrome P-450 dependent C–C bond cleavage: studies on 17α-hydroxylase-17,20-lyase

It is shown that formation of 17α-hydroxyandrost-5-en-3β-ol from pregnenolone occurs solely at the expense of the cleavage of the C-17–C-20 bond of the precursor and is best rationalised by invoking the participation of an FeIII–OOH intermediate in the acyl–carbon fission process.

Synthesis of 19-functionalised derivatives of 16α-hydroxy-testosterone: mechanistic studies on oestriol biosynthesis

The 19-hydroxy and 19-oxo derivatives of 16α,17β-dihydroxy-androst-4-en-3-one (9a) have been synthesised and shown to be intermediates in the biosynthesis of oestriol in the human placenta; O2 is incorporated into formic acid released from C-19 of the 19-oxo compound (9b).

Fluorescence Quenching Methods to Study Lipid‐Protein Interactions

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp‐containing intrinsic membrane protein with bromine‐containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine‐containing phospholipids from phospholipids containing carbon‐carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine‐containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.

The involvement of an O2-derived nucleophilic species in acyl–carbon cleavage, catalysed by cytochrome P-45017α: implications for related P-450 catalysed fragmentation reactions

The postulated iron peroxy species involved in acyl–carbon fission by 17α-hydroxylase-17,20-lyase (P-45017α) was trapped by an aldehyde analogue, partial 9, that was cleaved by the enzyme 10 times faster than the side-chain of the natural substrate 1.

Acyl-Carbon Bond Cleaving Cytochrome P450 Enzymes: CYP17A1, CYP19A1 and CYP51A1

Cytochrome P450 (P450 or CYP) enzymes in their resting state contain the heme-iron in a high-spin FeIII state. Binding of a substrate to a P450 enzyme allows transfer of the first electron, producing a Fe(II) species that reacts with oxygen to generate a low-spin iron superoxide intermediate (FeIII-O-O•) ready to accept the second electron to produce an iron peroxy anion intermediate (a, FeIII-O-O-). In classical monooxygenation reactions, the peroxy anion upon protonation fragments to form the reactive Compound I intermediate (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). However, when the substrate projects a carbonyl functionality, of the type b, at the active site as is the case for reactions catalyzed by CYP17A1, CYP19A1 and CYP51A1, the peroxy anion (FeIII-O-O-) is trapped, yielding a tetrahedral intermediate (c) that fragments to an acyl-carbon cleavage product (d plus an acid). Analogous acyl-carbon cleavage reactions are also catalyzed by certain hepatic P450s and CYP125A1 from Mycobacterium tuberculosis. A further improvisation on the theme is provided by aldehyde deformylases that convert long-chain aliphatic aldehydes to hydrocarbons. CYP17A1 is involved in the biosynthesis of corticoids as well as androgens. The flux toward these two classes of hormones seems to be regulated by cytochrome b 5, at the level of the acyl-carbon cleavage reaction. It is this regulation of CYP17A1 that provides a safety mechanism, ensuring that during corticoid biosynthesis, which requires 17α-hydroxylation by CYP17A1, androgen formation is avoided (Fig. 4.1).