Polyterpenoids as cholesterol and tetrahymanol surrogates in the ciliate Tetrahymena pyriformis.

The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols. The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect. In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T. pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids. Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids. No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability. Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T. pyriformis cells.

The effect of excess mevalonic acid on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

When T. pyriformis is grown in the presence of 10(-2)M-mevalonic acid, the uptake exceeds the cell's requirement for this biosynthetic intermediate. The majority of the excess mevalonic acid is diverted into ubiquinone-8 biosynthesis whereas the biosynthesis of tetrahymanol, the major product of the mevalonic acid pathway, is unchanged. In the presence of excess external mevalonic acid, the biosynthesis of mevalonic acid by the cell is inhibited. It is proposed that ubiquinone biosynthesis is normally regulated by mevalonic acid availability, whereas tetrahymanol biosynthesis is regulated primarily at a later point in the pathway.

Prokaryotic triterpenoids. 3. The biosynthesis of 2 beta-methylhopanoids and 3 beta-methylhopanoids of Methylobacterium organophilum and Acetobacter pasteurianus ssp. pasteurianus.

The incorporation of L-[methyl-3H,14C]methionine or L-(methyl-2H3)methionine into 2 beta-methyldiplopterol of Methylobacterium organophilum and various 3 beta-methylhopanoids of Acetobacter pasteurianus ssp. pasteurianus showed that all three hydrogen atoms of the transferred methyl group are retained in the triterpenoids. These methylations are compatible with a methylation substrate such as a delta 2-hopanoid in the case of the 2 beta-methylhopanoid biosynthesis and of a delta 2-hopanoid or squalene in the case of the formation of 3 beta-methylhopanoids. The intervention of intermediates possessing an exomethylene group or a cyclopropane ring is excluded.

The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.

The role of squalene synthetase in the inhibition of tetrahymanol biosynthesis by cholesterol in Tetrahymena pyriformis.

The biosynthesis of the triterpenoid alcohol tetrahymanol by Tetrahymena pyriformis is rapidly inhibited by the addition of cholesterol to the growth medium. The primary site of this inhibition by cholesterol has been established to be at the level of the enzyme squalene synthetase. The protein synthesis inhibitor cycloheximide produces an identical decline in squalene synthetase activity to that of cholesterol and the half-life of the enzyme is about 50 minutes. No direct inhibition of the enzyme is observed and suggests that cholesterol inhibits the actual synthesis of the enzyme squalene synthetase. Farnesol is accumulated during in vitro incubations derived from cells grown in the presence of cholesterol or cycloheximide.

Proposed pathway of triterpenoid carotenoid biosynthesis in Staphylococcus aureus: evidence from a study of mutants.

Mutants of Staphylococcus aureus were isolated which showed changes in pigment composition compared with the parent strain. On the basis of differences in their triterpenoid carotenoid composition they were classified into seven types. In five of these types, there appeared to be a blockage in the biosynthetic pathway which resulted in the absence of some products and accumulation of others. The changes in the other two types appeared to be a consequence of some change in regulation. A scheme for the biosynthesis of triterpenoid carotenoids is presented in which the first C30 intermediate, 4,4'-diapophytoene, is converted via 4,4'-diapophytofluene, 4,4'-diapo-zeta-carotene, 4,4'-diaponeurosporene, 4,4'-diaponeurosporenal, 4,4'-diaponeurosporenoic acid, and glucosyl-diaponeurosporenoate to the major pigment staphyloxanthin.

Non-specific biosynthesis of gammacerane derivatives by a cell-free system from the protozoon Tetrahymena pyriformis. Conformations of squalene, (3S)-squalene epoxide and (3R)-squalene epoxide during the cyclization.

1. A cell-free system from the protozoon Tetrahymena pyriformis was incubated with either [12-3H]squalene or (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene. Squalene was cyclized into tetrahymanol whereas racemic squalene epoxide was transformed into gammacerane-3 alpha,21 alpha-diol and gammacerane-3 beta,21 alpha-diol. After cyclization of (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene, both epimeric gammaceranediols were labelled with a tritium atom located at C-3, showing that no isomerization via a 3-oxo compound occurred. 2. The proton NMR spectra of the cyclization products of synthetic (2E, 22E)-(1,1,1,24,24,24-2H6)squalene and (RS)-(22E)-2,3-epoxy-2,3-dihydro-(1,1,1,24,24,24-2H6)squalene show that squalene and the (3S)enantiomer of its epoxide are cyclized in an all pre-chair conformation, whereas the (3R) enantiomer of squalene epoxide is cyclized in a pre-boat conformation as concerns the cycle A. 3. The squalene cyclase of T. pyriformis presents the same lack of substrate specificity as the cyclase of Acetobacter pasteurianum: in addition to squalene, its normal substrate, it also cyclizes both enantiomers of its epoxide. This conformational versatility is characteristic of squalene cyclases but no longer exists in the squalene epoxide cyclases from eukaryotes.

Non-specific lanosterol and hopanoid biosynthesis be a cell-free system from the bacterium Methylococcus capsulatus.

1. A cell-free system from the bacterium Methylococcus capsulatus was incubated with [12-3H]-squalene; diploptene and diplopterol, normally present in the bacterium, were labelled. 2 The same cell-free system was incubated with (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene. Several radioactive 3-hydroxytriterpenes were purifed. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3-epilanosterol. In addition, radioactive 3 alpha-hydroxy and 3 beta-hydroxydiploptene were formed. 3. These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i.e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3-hydroxyhopanoids. 4. The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3 beta-hydroxy 4 alpha-methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3-epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3-oxo compound. Therefore the squalene expoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate-specific as it can also cyclize the (3R) enantiomer into 3-epilanosterol.

Non-specific biosynthesis of hopane triterpenes by a cell-free system from Acetobacter pasteurianum.

1. A cell-free system from the bacterium Acetobacter pasteurianum was incubated with [12-3H]squalene; diploptene and diplopterol, hopanoids normally present in the bacterium, were labelled. Their radioactivity was confirmed by purification using thin-layer chromatography, synthesis of derivatives and recrystallization to constant specific activity. This demonstrates the direct cyclization of squalene into diploptene and diplopterol, catalysed by a squalene cyclase activity in A. pasteurianum. 2. The same cell-free system transformed (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene into labelled 3 alpha-hydroxyhop-22(29)-ene, 3 beta-hydroxyhop-22(29)-ene, hopane-3 alpha,22-diol and hopane-3 beta,22-diol. Their radioactivity was similarly confirmed. This bacterial homogenate is thus capable of cyclizing an unnatural substrate, 2,3-epoxy-squalene, into 3-hydroxyhopanoids normally absent in the bacterium. 3. The 3 alpha-hydroxy and 3 beta-hydroxyhopanoids could have been enzymatically interconverted via the 3-oxo compound. Synthetic racemic (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene was incubated and gave rise to 3-3H-labelled 3 alpha and 3 beta-hydroxyhopanoids. This excludes an isomerization via a 3-oxo compound which would give unlabelled 3-hydroxyhopanoids. 4. In conclusion, the cyclase of A. pasteurianum accepts the replacement of the normal substrate, squalene, by the corresponding epoxide. Furthermore it is not selective in the stereochemistry of the epoxide and cyclizes both enantiomers, contrary to the epoxysqualene cyclase of eukaryotes.

2,3-Oxidosqualene cyclase and cycloartenol-s-adenosylmethionine methyltransferase activities in vivo in the cotyledon and axis tissues of germinating pea seeds.

Axis tissues, root and shoot, of germinating pea seedlings actively synthesize sterol from [2-14C]mevalonate during the first 3 days of germination. In addition to the intermediates of sterol synthesis, cycloartenol and 24-methylenecycloartanol, these tissues also form the triterpene beta-amyrin. The cyclase catalysing the formation of cycloartenol from oxidosqualene is about four times as active as that for beta-amyrin synthesis. 2. Sterol synthesis in the cotyledon is negligible, but cycloartenol and 24-methylenecycloartanol, as well as beta-amyrin, are synthesized there. Oxidosqualene cyclase activity in this tissue is 2.6 times as active for beta-amyrin synthesis as for cycloartenol synthesis. 3. Comparison of the relative amounts of 14C in cycloartenol and 24-methylenecycloartanol in the axis tissues and cotyledons of 3-day-old seedlings point to relatively active cycloartenol-S-adenosylmethionine methyltransferase systems in both axis tissues and a poorly active system in the cotyledon. 4. The role of beta-amyrin synthesis in the germinating pea seedling is discussed.

Studies on the biosynthesis of tetrahymanol in Tetrahymena pyriformis. The mechanism of inhibition by cholesterol.

Tetrahymanol biosynthesis by the protozoan Tetrahymena pyriformis was progressively inhibited by the inclusion of cholesterol in the growth medium. Studies with labelled precursors of tetrahymanol have established that there are two major sites of inhibition in whole cells. The inhibition at the first site, between acetate and mevalonate, occurred rapidly after addition of cholesterol. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), a predominantly cytosolic enzyme in this organism, was not inhibited in cholesterol-grown cells nor by addition of cholesterol directly to the assay medium. The second major site of inhibition in whole cells is between mevalonate and squalene and this is accompanied by inhibition of the enzyme that converts farnesyl-pyrophosphate into squalene (squalene synthetase). Squalene cyclase is partially inhibited. The conversion of mevalonate into tetrahymanol in vitro was not inhibited by the addition of cholesterol to the assay medium. Tetrahymanol added to the culture medium is taken up by the cells but does not inhibit endogenous biosynthesis. It is suggested that cholesterol inhibits the later stages of tetrahymanol biosynthesis by causing a change in membrane structure and function which alters the activity of membrane-bound enzymes.