The origin of the 14α-hydrogen in cholesterol biosynthesis

Akhtar, Muhammad; Rahimtula, Anver D.; Watkinson, I. A.; Wilton, David C.; Munday, K. A.

doi:10.1039/c29690000149

Cited by 31 publications

(38 citation statements)

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“…This change in reaction mechanism is accompanied by a change of His-117 to a glutamic acid residue. As noted by Akhtar et al [84], carbon-carbon double-bond reduction in steroids can occur by mechanisms that involve either protonation or hydride transfer occurring first. Protonation would give rise to a carbonium-ion intermediate, so that hydride addition could occur by a Markovnikov addition.…”

Section: Figure 4 Akr Active Sitementioning

confidence: 94%

Comparative anatomy of the aldo–keto reductase superfamily

Jez

Bennett

Schlegel

et al. 1997

Biochemical Journal

569

588

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The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.

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Section: Figure 4 Akr Active Sitementioning

confidence: 94%

Comparative anatomy of the aldo–keto reductase superfamily

Jez

Bennett

Schlegel

et al. 1997

Biochemical Journal

569

588

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“…The incubation of cholest-7-en-3j-01 with rat liver enzymes in tritiated water would yield under these conditions cholesterol labelled at positions 3a [15], 7a,8P [16] and possibly, if isomerization takes place, at position 9a, each position containing different amounts of tritium. Therefore the localization and the quantitative evaluation of radioactivity would be very laborious.…”

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confidence: 99%

The Reversibility of the Isomerization of the Δ⁸ to Δ⁷ Bond in Cholesterol Biosynthesis

Scala

Galli-Kienle

Anastasia

et al. 1974

European Journal of Biochemistry

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The reversibility of the isomerization of the A s to A7 bond in the biosynthesis of cholesterol has been re-examined. For this purpose [4-'4C,9a-3H,]cholest-7-en-3~-ol has been incubated with the 10000 x g soluble fraction of rat liver homogenate both under anaerobic and aerobic conditions. Almost complete exchange of the 9a hydrogen with the medium has been observed under anaerobic conditions, whereas under aerobic conditions 34 % exchange with the medium has been calculated from the 3H/14C ratio found in cholesterol isolated after incubation. The stereospecificity of the A7 to As isomerization has been established by incubating in analogous conditions [4-14C,7-3Hl]cholest-7-en-3P-01. The synthesis of each labelled substrate is described.Cholesterol biosynthesis involves the isomerization of the double bond at position 8 of lanosterol to the 7 position. The reaction may occur at any step of the biosynthesis after the removal of the methyl group at C-32 [l]. The isomerization implies the stereospecific removal of the 7P hydrogen and the addition of a solvent hydrogen at the 9a positionThe possible reversibility of this reaction has been considered and different authors [l ,4] have presented evidence that no detectable amounts of cholest-8-en-3P-01 are formed by incubating cholest-7-en-3~-01 with a microsomal preparation of rat liver. On the other hand it has been recently demonstrated that tritium from the medium is incorporated at the 9a position of cholest-7-en-3/3-01, when this sterol is incubated either with microsomes or with the 10000 x g soluble fraction of rat liver homogenate under anaerobic conditions [5]. On the basis of these results it has been concluded that the biological conversion of cholest-8-en-3P-01 to cholest-7-en-38-01 is a reversible process. However under physiological conditions cholest-7-en-3/l-ol is efficiently converted to cholesterol and it does not accumulate in the same way as under the anaerobic conditions utilized in the above-mentioned experiments. It was of interest therefore to consider the reversibility of the isomerization under aerobic conditions. For this purpose [4-14C,9a-3H1]cholest-7-en3p-01 was synthesized and incubated with the 10 000 x g soluble fraction of rat liver homogenate under aerobic ~3 1 .conditions. The decreased 3H/14C ratio found in cholesterol derived from the incubations demonstrated that isomerization from cholest-7-en-3P-01 to cholest-8-en-3P-01 takes place also during the biological conversion of the former to cholesterol. The stereospecificity of the reaction is also demonstrated under both aerobic an anaerobic conditions. MATERIALS AND METHODS MaterialsTritiated water and tritiated sodium boron hydride were obtained from Amersham (U.K.) ; N,O-bis(trimethylsily1)-trifluoroacetamide from Pierce Chemicals; osmium tetroxide from Merck ; cholesta-5,7-dien-38-01 acetate from Fluka A.G. Radioactive Thin-Layer Chromatography ScanningThis was carried out using a Packard radioactivechromatogram scanning system, model 7201 and silica gel H F layers (0.25 mm ...

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“…Radioactive dimethyl-8,14-diene (IIIa) used in the previous studies (Akhtar et al 1969a;Canonica et al 1968b) was labelled with tritium at undetermined positions, some of which are expected to be labilized during cholesterol biosynthesis, therefore not allowing the true estimation of the overall conversion to be made. Thus for a critical evaluation of the problem several specifically labelled steroids were required.…”

Section: Resultsmentioning

confidence: 99%

“…dimethylcholesta-8,14-dien-3fl-ol (IIIa, hereinafter called dimethyl-8,14-diene) into cholesterol has been demonstrated (Akhtar et al 1969a;Canonica et al 1968b). This paper provides evidence for the obligatory formation of dimethyl-8,14-diene (IIIa) from [3ac-3H]dihydrolanosterol (Ia) and describes experiments pertinent to the mechanism for the further conversion of the compound (IlIa) into cholesterol.…”

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confidence: 96%

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

Watkinson

Wilton

Munday

et al. 1971

Biochemical Journal

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It was shown that 100mug quantities of 4,4'-dimethyl[2-(3)H(2)]cholesta-8,14-dien-3beta-ol (IIIa), tritiated cholesta-8,14-dien-3beta-ol, 4,4'-dimethyl[2-(3)H(2)]cholesta-7,14-dien-3beta-ol, dihydro[2-(3)H(2)]lanosterol and [24-(3)H]lanosterol were converted by a 10000g supernatant of rat liver homogenate into cholesterol in 17%, 54%, 6%, 9.5% and 24% yields respectively. From an incubation of dihydro[3alpha-(3)H]lanosterol with a rat liver homogenate in the presence of a trap up to 38% of the radioactivity was found to be associated with a fraction that was unambiguously shown to be 4,4'-dimethylcholesta-8,14-dien-3beta-ol. Another related compound, 4,4'-dimethylcholesta-7,14-dien-3beta-ol was also shown to be equally effective in its ability to trap compound (IIIa) from an incubation of dihydro[3alpha-(3)H]lanosterol. The mechanism of the further conversion of the compound (IIIa) into cholesterol occurred by the reduction of the 14,15-double bond and involved the addition of a hydrogen atom from the medium to C-15 and another from the 4-position of NADPH to C-14. Two possible mechanisms for the removal of the 14alpha-methyl group in sterol biosynthesis are discussed.

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The origin of the 14α-hydrogen in cholesterol biosynthesis

Cited by 31 publications

References 1 publication

Comparative anatomy of the aldo–keto reductase superfamily

Comparative anatomy of the aldo–keto reductase superfamily

The Reversibility of the Isomerization of the Δ⁸ to Δ⁷ Bond in Cholesterol Biosynthesis

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

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The origin of the 14α-hydrogen in cholesterol biosynthesis

Cited by 31 publications

References 1 publication

Comparative anatomy of the aldo–keto reductase superfamily

Comparative anatomy of the aldo–keto reductase superfamily

The Reversibility of the Isomerization of the Δ8 to Δ7 Bond in Cholesterol Biosynthesis

The formation and reduction of the 14,15-double bond in cholesterol biosynthesis

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The Reversibility of the Isomerization of the Δ⁸ to Δ⁷ Bond in Cholesterol Biosynthesis