Farnesyl pyrophosphate synthetase from Bacillus subtilis.

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.

Inhibition of liver prenyltransferase by alkyl phosphonates and phosphonophosphates.

n-Pentyl and n-decyl phosphonate and the corresponding phosphonophosphates were found to inhibit cholesterol synthesis from mevalonate in the 10000 X g supernatants of liver homogenates and the synthesis of farnesyl pyrophosphate from geranyl and isopentenyl pyrophosphate by purified liver prenyltransferase. Kinetic analysis of the inhibition of prenyltransferase showed that the phosphonates and the phosphonophosphates interacted with two forms, or two sites, of the enzyme. The order of increasing potency was C5-phosphonate less than C10-phosphonate less than C5-phosphonophosphate less than C10-phosphonophosphate. The phosphonophosphates were at least ten times stronger inhibitors than the phosphonates.

Abnormal induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in leukocytes from subjects with heterozygous familial hypercholesterolemia.

Human leukocytes isolated from fresh defibinated blood were shown to utilize acetate and mevalonate for sterol synthesis. The capacity of the leukocytes to synthesize sterols is limited severely as compared to their ability to convert mevalonate into farnesyl pyrophosphate (which they hydrolyze rapidly to free farnesol) and into squalene. When leukocytes are incubated in a medium containing lipid-free serum, synthesis of sterols from acetate, but not from mevalonate, is much enhanced. It was shown that this increased synthesis resulted from increased levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in the cells. A comparison was made of the activation of sterol synthesis from acetate in leukocytes of normal individuals and of heterozygous familial hypercholesterolemics. The latter group responded to incubation in lipid-free sera with a significantly higher activation than the cells of normocholesterolemics. This activation was shown to be well correlated with a higher induction of 3-hydroxy-3-methylglutaryl-CoA reductase in the heterozygous cells than in the normals. The leukocytes of a heterozygous familial hypercholesterolemic individual were found to release, into a lipid-free incubation medium, more endogenously synthesized [3H]sterol (but not [3H]squalene) than the cells of a normal person. It is suggested that the genetic abnormality in heterozygous familial hypercholesterolemia could be accounted for by a mutation resulting in a weaker binding of a sterol repressor by heterozygous cells than by normal cells.

Biosynthesis of trans, trans- and cis, trans-farnesols by soluble enzymes from tissue cultures of Andrographis paniculata.

A cell-free system obtained from tissue cultures of Andrographis paniculata produces 2-trans,6-trans-farnesol (trans,trans-farnesol) and 2-cis,6-trans-farnesol (cis,trans-farnesol) (5:1), incorporating 10% of the radioactivity from 3R-[2-(14)C]mevalonate. There is total loss of (3)H from 3RS-[2-(14)C,(4S)-4-(3)H(1)]mevalonate and total retention from the (4R) isomer in both the trans,trans-farnesol and cis,trans-farnesol formed. When 3RS-[2-(14)C,5-(3)H(2)]mevalonate is used as substrate, there is total retention of (3)H in the trans,trans-farnesol, but loss of one-sixth of the (3)H in the cis,trans-farnesol. With (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-trans,trans -farnesol and (1R)- and (1S)-[4,8,12-(14)C(3),1-(3)H(1)]-cis, trans-farnesol as substrates, the label is lost from the (1R)-cis,trans and (1S)-trans,trans isomers but retained in the (1R)-trans,trans and (1S)-cis,trans isomers; this shows that the pro-1S hydrogen is exchanged in the conversion of trans,trans-farnesol into cis,trans-farnesol and the pro-1R hydrogen in the conversion of cis,trans-farnesol into trans,trans-farnesol. (1R)-[1-(3)H(1)]-trans,trans-Farnesol and (1R)-[1-(3)H(1)]-cis,trans-farnesol have been synthesized by asymmetric chemical synthesis and exchanged with liver alcohol dehydrogenase. Both the trans- and the cis-alcohol exchange the pro-1R hydrogen atom.

Some studies on the biosynthesis of ubiguinone, isoprenoid alcohols, squalene and sterols by marine invertebrates.

The ability of fourteen marine invertebrates to utilize [(14)C]mevalonate for the biosynthesis of isoprenoid compounds was investigated. Several of the animals, in particular crustaceans, bivalve molluscs, a coelenterate and a sponge, were unable to synthesize squalene and sterols, whereas gastropod molluscs, echinoderms, an annelid and a sponge could. Regardless of sterol-synthesizing ability the animals (with the exception of a sponge) always made dolichol and ubiquinone, and thus a specific block in squalene and sterol synthesis was indicated in some animals. Radioactivity accumulated in relatively large amounts in farnesol and geranylgeraniol in those animals incapable of making sterols.

Synthesis of 10,11-dihydrofarnesyl pyrophosphate from 6,7-dihydrogeranyl pyrophosphate by prenyltransferase.

The syntheses of 6,7-dihydrogeraniol and of its pyrophosphate are described. It is shown that this analogue of geranyl pyrophosphate is a substrate for liver prenyltransferase and that the product synthesized by this enzyme from it and isopentenyl pyrophosphate is 10,11-dihydrofarnesyl pyrophosphate. The K(m) value for 6,7-dihydrogeranyl pyrophosphate was determined to be 1.11+/-0.19mum as compared with 4.34+/-1.71mum for geranyl pyrophosphate. The maximum reaction velocity with the artifical substrate was, however, only about one-fourth of that observed with geranyl pyrophosphate. The binding of isopentenyl pyrophosphate to the enzyme was not affected by the artificial substrate.

Isoprenoid biosynthesis in a cell-free system from pea shoots.

A cell-free system consisting of a soluble fraction and plastid fragments from pea shoots incorporates 2-(14)C-mevalonate very actively into farnesol, squalene, geranylgeraniol, and other isoprenoids of different carbon-chain lengths. The products have different cofactor requirements, which makes it possible to channel the pathway into different products by varying the incubation mixture.