Studies on the mechanism and regulation of C-4 demethylation in cholesterol biosynthesis. The role of adenosine 3′:5′-cyclic monophosphate

1. An assay for demethylation has been developed based on the release of tritium from 4,4-dimethyl[3alpha-(3)H]cholest-7-en-3beta-ol (II). 2. The maximum release of (3)H from 3alpha-(3)H-labelled compound (II) in a rat liver microsomal preparation occurs in the presence of NADPH and NAD(+) under aerobic conditions. 3. Incubation of 3alpha-(3)H-labelled compound (II) with NADPH under aerobic conditions leads to the formation of a 3alpha-(3)H-labelled C-4 carboxylic acid. This compound undergoes dehydrogenation on subsequent anaerobic incubation with NAD(+). 4. The (3)H released from the steroid was located in [4-(3)H]nicotinamide and the medium. Incubation with synthetic [4-(3)H(2)]NADH gave a similar result. 5. In the presence of glutamate dehydrogenase and alpha-oxoglutarate part of the (3)H released from the steroid was transferred to glutamate. 6. A series of 3-oxo steroids were reduced equally well by [4-(3)H(2)]NADH and [4-(3)H(2)]NADPH. The reduction of 5alpha-cholest-7-en-3-one was shown to use the 4B H atom from the nucleotide. 7. 3':5'-Cyclic AMP was shown to be a competitive inhibitor of the 3beta-hydroxy dehydrogenase enzyme in the demethylation reaction.     

Mechanistic studies of lanosterol C-32 demethylation. Conditions which promote oxysterol intermediate accumulation during the demethylation process

Conditions have been established which promote the accumulation of the dihydrolanosterol C-32 demethylation intermediates lanost-8-en-3 beta,32-diol and 3 beta-hydroxylanost-8-en-32-aldehyde with intact hepatic microsomes. Accumulation of dihydrolanosterol-derived oxysterols occurs with a variety of assay manipulations which include short incubation times, limiting enzyme amounts, high pH, and increasing substrate concentration. In addition, competitive inhibition of dihydrolanosterol demethylation by lanosterol, or the reciprocal inhibition of lanosterol demethylation by dihydrolanosterol, leads to oxysterol accumulation at the expense of demethylated end product. Similarly, the nonsteroidal demethylase inhibitors miconazole and ketoconazole promote oxysterol accumulation in a concentration-dependent manner. Finally, cholesterol loading of isolated microsomes results in changes in the measured kinetic constants, Km and Vmax, and results in enhanced oxysterol accumulation above that seen in control microsomal preparations. The major oxysterol intermediate accumulated under all the conditions described above is the C-32 aldehyde in an approximate 3:1 ratio to the C-32 alcohol. These data support the conclusion that a single enzyme species is responsible for all three oxidations of the C-32 demethylation sequence. In addition, intermediates which do not routinely accumulate during demethylation are freely diffusible from the enzyme when appropriate conditions are established to prevent their further metabolism.     

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.     

Decarboxylation of uroporphyrinogen III by erythrocyte uroporphyrinogen decarboxylase. Evidence for a random decarboxylation mechanism.

The isomeric composition of type-III heptacarboxylic porphyrinogens derived from decarbosylation of uroporphyrinogen III by erythrocyte uroporphyringogen decarboxylase was analysed by h.p.l.c. with electrochemical detection. All four possible isomers were identified, and there were little differences in the proportion of isomers formed by erythrocytes from normal subjects and from patients with sporadic porphyria cutanea tarda. The results provide conclusive evidence that the normal decarboxylation pathway is random in nature, and the fourth isomer only increases when enzyme abnormality is found.     

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.     

Evidence for a water-soluble intermediate in exchange of cholesterol between membranes

The mechanism of inter-membrane cholesterol exchange has been a matter of some debate. Evidence from kinetic studies indicates that cholesterol must transfer to and from membranes in a water-soluble form. In this study attempts have been made to demonstrate that this occurs using either dialysis membranes or a barrierless multiphase polymer system to physically separate the membranes. In both systems small amounts of cholesterol were seen to transfer from one membrane pool to another using both liposomes and erythrocyte membranes as donors or acceptors. The cholesterol transfer was shown to be independent of the movement of other membrane components. The amount of transfer observed was limited by the physical properties of the systems employed. The barrier to cholesterol transfer in the dialysis membrane system is primarily the pore size of the membrane, while in the multiphase polymer system the transfer was limited by the viscosity of the medium and the distance between the lower and upper phases containing the membranes. Nevertheless, the results provide evidence that cholesterol transfer is by a dissociation of molecules from membranes into the aqueous medium and does not require the formation of a collision complex between the membranes.     

Evidence for an intermediate in quinolinate biosynthesis in Escherichia coli.

Evidence for the formation of an unstable intermediate in the synthesis of quinolinate from aspartate and dihydroxyacetone phosphate by Escherichia coli was obtained using toluenized cells of nadA and nadB mutants of this organism and partially purified A and B proteins in dialysis and membrane cone experiments. The results of these experiments indicate that the nadB gene product forms an unstable compound from aspartate in the presence of flavine adenine dinucleotide, and that this compound is then condensed with dihydroxyacetone phosphate to form quinolinate in a reaction catalyzed by the nadA gene product.     

Dissecting the sterol C-4 demethylation process in higher plants. From structures and genes to catalytic mechanism

Sterols become functional only after removal of the two methyl groups at C-4. This review focuses on the sterol C-4 demethylation process in higher plants. An intriguing aspect in the removal of the two C-4 methyl groups of sterol precursors in plants is that it does not occur consecutively as it does in yeast and animals, but is interrupted by several enzymatic steps. Each C-4 demethylation step involves the sequential participation of three individual enzymatic reactions including a sterol methyl oxidase (SMO), a 3β-hydroxysteroid-dehydrogenase/C4-decarboxylase (3βHSD/D) and a 3-ketosteroid reductase (SR). The distant location of the two C-4 demethylations in the sterol pathway requires distinct SMOs with respective substrate specificity. Combination of genetic and molecular enzymological approaches allowed a thorough identification and functional characterization of two distinct families of SMOs genes and two 3βHSD/D genes. For the latter, these studies provided the first molecularly and functionally characterized HSDs from a short chain dehydrogenase/reductase family in plants, and the first data on 3-D molecular interactions of an enzyme of the postoxidosqualene cyclase sterol biosynthetic pathway with its substrate in animals, yeast and higher plants. Characterization of these three new components involved in C-4 demethylation participates to the completion of the molecular inventory of sterol synthesis in higher plants.     

The mechanism of alkylation at C-24 during clionasterol biosynthesis in Monodus subterraneus

Clionasterol isolated from Monodus subterraneus grown in the presence of methionine-[methyl-²H₃] contained four ²H atoms showing the participation of a 24-ethylidene sterol intermediate in its biosynthesis. Clionasterol isolated from M. subterraneus grown in the presence of mevalonic acid-[2-¹⁴C,(4R)-4-³H₁ had a ¹⁴C:₃H atomic ratio of 5:3 indicating that the 24-ethylidene sterol intermediate is reduced directly to clionasterol and not isomerized to a Δ²⁴-sterol which is then reduced.     

Proposed mechanism for the bacterial bioluminescence reaction involving a dioxirane intermediate

We propose here a verifiable mechanism for the bacterial bioluminescence reaction involving a dioxirane intermediate. Participation of the dioxirane predicts either formation of an excited carbonyl, rather than the flavin, as the primary excited state in the reaction, or, through a CIEEL mechanism, the C4a hydroxyflavin or the chromophore of a secondary emitter protein could become excited. We propose energy transfer from the primary excited state to the C4a hydroxyflavin in the absence of the lumazine protein or the yellow fluorescence protein, while in the presence of either of the secondary emitter proteins, excitation energy would be transferred to the second protein-bound chromophore. The mechanism is similar to other currently discussed mechanisms, except in the final steps leading to the primary excited state. The mechanism is consistent with the known details of the reactions of dioxiranes and of flavins and with recent studies of the secondary emitter proteins and bacterial luciferases.     

The stereochemistry of hydrogen elimination from C-7 in cholesterol and ergosterol biosynthesis

The synthesis of [7alpha-(3)H]lanosterol is described. It is shown that in the conversion of [7alpha-(3)H,26,27-(14)C(2)]lanosterol into cholesterol by a rat liver system, it is the 7beta-hydrogen atom that is predominantly removed. On the other hand, the conversion of doubly labelled lanosterol into ergosterol by whole yeast cells results in the loss of the 7alpha-hydrogen atom. These results therefore suggest that the C-7 hydrogen atoms with opposite stereochemistry are labilized by the rat liver and the yeast Delta(8)-Delta(7) steroid isomerases.     

Evidence for a peroxide-initiated free radical mechanism of prostaglandin biosynthesis

High levels of NaCN (20 to 250 mM) were required to inhibit cyclooxygenase catalysis and cause extended lag periods (up to 1.6 min), whereas CO failed to inhibit catalysis. This NaCN inhibition was easily overcome by endogenous or exogenous hydroperoxides. Added hydroperoxides acted to eliminate lag periods without undergoing net conversion to other chemical species. In addition, experiments with glutathione peroxidase inhibition showed that hydroperoxides were essential not only in the early phases, but throughout catalysis. In spectrophotometric experiments, NaCN formed a complex with ferriheme cyclooxygenase (Kd = 1.3 mM) and inhibited hydroperoxide interaction with this form of the enzyme. Phenolic antioxidants, only slightly extended lag periods while inhibiting oxygenation rates more than 50%. Low levels of phenol (which is normally stimulatory) or alpha-naphthol when combined with NaCN or glutathione peroxidase (agents which interfere with peroxide activation) resulted in potent synergistic inhibition with long lag times. A mechanism consistent with all of the above properties of cyclooxygenase has been elucidated, Further mechanistic explanation was sought for reaction-catalyzed self-inactivation of cyclooxygenase. This phenomenon could not be explained simply by heme lability, or cyclooxygenase sensitivity to destruction by ambient hydroperoxides, Rather, it appears to involve a destructive reaction intermediate intrinsic to involve a destructive reaction intermediate intrinsic to the cyclooxygenase mechanism.     

Stereochemistry of C-4 demethylation during conversion of obtusifoliol into poriferasterol by Ochromonas malhamensis

Obtusifoliol-[2,2,4-³H₃] was synthesised and incubated with the chrysophyte alga Ochromonas malhamensis which converted it into poriferasterol. A reaction sequence applied to poriferasterol showed that the tritium retained at C-4 occupied the 4α-position. This demonstrates that biological C-4 demethylation of a 4α-methylsterol precursor by O. malhamensis results in the axial 4β-hydrogen being inverted into the equatorial 4α-position of the 4-desmethyl sterol product.