Mevalonic acid phosphorylation

When correspondingly the biosynthetic pathway to terpenes is tracked, three carbons in dimethylaUyl diphosphate carry the label. First, two molecules of acetyl-CoA condense to give acetoacetyl-CoA. Then an enzyme-bound acetyl residue is transferred in the manner of an aldol addition, and reduction with NADPH finally produces (R)-mevalonic acid. Phosphorylation by ATP, dehy-drative decarboxylation and isomerisation of the isopentenyl diphosphate lead to dimethylaUyl diphosphate. 

In keeping with its biogenetic origin m three molecules of acetic acid mevalonic acid has six carbon atoms The conversion of mevalonate to isopentenyl pyrophosphate involves loss of the extra carbon as carbon dioxide First the alcohol hydroxyl groups of mevalonate are converted to phosphate ester functions—they are enzymatically phosphorylated with introduction of a simple phosphate at the tertiary site and a pyrophosphate at the primary site Decarboxylation m concert with loss of the terti ary phosphate introduces a carbon-carbon double bond and gives isopentenyl pyrophos phate the fundamental building block for formation of isoprenoid natural products... 

The principal steps in the mechanism of polyisoprene formation in plants are known and should help to improve the natural production of hydrocarbons. Mevalonic acid, a key intermediate derived from plant carbohydrate via acetylcoen2yme A, is transformed into isopentenyl pyrophosphate (IPP) via phosphorylation, dehydration, and decarboxylation (see Alkaloids). IPP then rearranges to dimethylaHyl pyrophosphate (DMAPP). DMAPP and... 

Mevalonic acid is then modified by phosphorylation and decarboxylation, and several molecules of it are condensed to form cholesterol in a complex series of eight reactions. 

Figure 8-6. Hormonal regulation of cholesterol synthesis by reversible phosphorylation of HMG CoA reductase. Availability of mevalonic acid as the fundamental building block of the sterol ring system controls flux through the pathway that follows. cAMP, cyclic adenosine monophosphate HMG CoA, hydroxymethylglutary I CoA.

Steroids are members of a large class of lipid compounds called terpenes. Using acetate as a starting material, a variety of organisms produce terpenes by essentially Lire same biosynlheLic scheme (Fig. 3). The sell-condensation of two molecules of acetyl coenzyme A (CoA) forms acetoacetyl CoA. Condensation of acetoacetyl CoA with a third molecule of acetyl CoA, then followed by an NADPH-mediated reduction of the thioester moiety produces mevalonic acid (22). Phosphorylation of (22) followed by concomitant decarboxylation and dehydration processes... 

Formation of the biological isoprene unit from mevalonic acid has been shown to proceed by stepwise phosphorylation of both alcohol groups, then elimination and decarboxylation to yield 3-methyl-3-butenyl pyrophosphate, 9 (often called A3-isopentenyl pyrophosphate) ... 

The classic route for the formation of the C5 building blocks of terpenoid bios)mthesis in plants is via the reactions of the mevaionate pathway, first demonstrated in yeast and mammals. This well-characterized sequence (Fig. 5.3) involves the stepwise condensation of three molecules of acetyl coenz)mie A (AcCoA) to form the branched C6 compound, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Following the reduction of HMG-CoA to mevalonic acid, two successive phosphorylations and a decarboxylationelimination yield the C5 compound, IFF. 

It is believed that the function of ATP is to phosphorylate mevalonic acid pyrophosphate at the 3-position. 

Mevalonic acid is phosphorylated and decarboxylated to form the 5-carbon (C-5) isoprene compound, isopentenyl pyrophosphate. 

Most successful attempts to isolate the enzymes involved in terpene biosynthesis have come from these early stages. Crude in vitro systems will frequently convert - [2- C]mevalonic acid (1) into its phosphate (2) and pyrophosphate (3), isopentenyl pyrophosphate (4), and dimethylallyl pyrophosphate (5). However, only traces of radioactivity are recovered from the prenol pyrophosphates (6). As well as phosphorylating mevalonic acid the same enzyme, or a related one, is... 

Mevalonic acid was discovered by Folker s group at Merck, Sharpe, and Dohme. The initial isolation was based upon the fact that it acted as a growth factor, or vitamin, for a strain of bacteria [35]. Once the structure had been determined, it was apparent that the molecule might well be the isoprenoid precursor that had been sought for many years. Subsequent experiments demonstrated that the sole (or nearly so) fate of the molecule was polyisoprenoid synthesis. In examining the role of cofactors necessary for the synthesis of cholesterol from mevalonate, only ATP and NADPH were found to be required. Experiments with a solubilized preparation from yeast demonstrated that there were 3 phosphorylated intermediates that could be isolated. These were shown to be mevalonic-5-phosphate, mevalonic-5-pyrophos-phate, and isopentenyl pyrophosphate [9]. These intermediates are derived from mevalonate in a sequence of phosphorylations, and the enzymes for all reactions have been obtained in homogeneous form. These enzymes, as well as the rest that lead to the synthesis of famesyl pyrophosphate, are cytosolic proteins. 

Figure 3.6 illustrates how, through phosphorylation, elimination and decarboxylation, mevalonic acid is converted to isopentenyl pyrophosphate, which can be isomerised enzymically to pentenyl pyrophosphate. 

From these and many similar examples it became evident that discrimination between enantiomers is often a matter of degree. Absolute discrimination, however, is shown by specific oxidases like D-amino acid oxidase of mammalian kidney and L-amino acid oxidase of snake venom. "No one [member] of this class of biological catalysts has yet been known to attack measurably an amino acid antipodal to its normally susceptible category of substracts ) [Greenstein and Winitz (1961)] [Zellor and Maritz (1945)]. Equally selective is the phosphorylation of mevalonic acid by the enzyme mevalonic kinase the R- form is phosphorylated, the S- form is unaffected (Tchen 1958). 

Steroids are members of a large class of lipid compounds called terpenes. Using acetate as a starting material, a variety of organisms produce terpenes by essentially the same biosynthetic scheme (Fig. 8). The self-condensation of two molecules of acetyl coenzyme A (CoA) forms acetoacetyl CoA. Condensation of acetoacetyl CoA with a third molecule of acetyl CoA, then followed by an NADPH-mediated reduction of the thioester moiety produces mevalonic acid [150-97-0] (72). Phosphorylation of (72) followed by concomitant decarboxylation and dehydration processes produce isopentenyl pyrophosphate. Isopentenyl pyrophosphate isomerase establishes an equilibrium between isopentenyl pyrophosphate and 3,3-dimethylallyl pyrophosphate (73). The head-to-tail addition of these isoprene units forms geranyl pyrophosphate. The addition of another isopentenyl pyrophosphate unit results in the sesquiterpene (C15) famesyl pyrophosphate (74). Both of these head-to-tail additions are catalyzed by prenyl transferase. Squalene synthetase catalyzes the head-to-head addition of two achiral molecules of famesyl pyrophosphate, through a chiral cyclopropane intermediate, to form the achiral triterpene, squalene (75). 

The mechanism for converting mevalonic acid into mevaionyi phosphate is essentially an Sn2 reaction with an adenosyl pyrophosphate leaving group (Section 27.3). A second Sn2 reaction converts mevaionyi phosphate to mevaionyi pyrophosphate. ATP is an excellent phosphorylating reagent for nucleophiles because its phosphoanhydride bonds are easily broken. The reason that phosphoanhydride bonds are so easily broken is discussed in Section 27.4. 

Mevalonic acid is activated by phosphorylation in the biosynthesis of polyisoprene ... 

Nature uses phosphate and pyrophosphate as good leaving groups, just as organic chemists use sulfonate esters and halides as good leaving groups. In the next step of this biosynthesis, mevalonic acid is phosphorylated ... 

Mevalonic acid is the precursor of the terpenes. Write its structure. After phosphorylation, it undergoes a decarboxylative elimination to yield 3-isopentenyl pyrophosphate. Outline the mechanism of this reaction. 

Mevalonate kinase (EC 2.7.1.36) phosphorylates mevalonic acid, the NADPH-reduced form of HMG-CoA. The reaction is ATP and Mg dependent. The enzyme was purified from a C. roseus cell suspension culture, in which, after induction a specific activity of 1.5 nkat/mg protein is found [12]. The purification protocol comprised ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. By gel filtration an M,. of 105,000 was found for mevalonate kinase. 

The first specific precursor for terpenoids in the cytoplasma is the Cg molecule mevalonic acid (MVA), which is built via the classical acetate/mevalonate pathway and converted by a series of phosphorylating and decarboxylation reactions into C5 isopentenyldiphosphate (IPP), the universal building block for chain elongation up to C20. In the chloroplasts, the biosynthesis of IPP starts from glyceraldehyde-3-phosphate and pyruvate to give l-deoxy-D-xylulose-5-phosphate (DOXP) via the non-mevalonate pathway as a recently detected alternative IPP route [19]. The reaction is catalyzed by the enzyme DOXP synthase and can be inhibited by a breakdown product of the herbicide clomazone [12]. 

Dolichol phosphate, which is a crucial intermediate, has been shown to be synthesised in a variety of tissues and subcellular fractions, of which mitochondrial outer membranes are especially active. These are also a major store of dolichol. The synthesis can start from isopentenyl pyrophosphate or its precursor, mevalonic acid (see the reviews of Beytia and Porter, 1976 Daleo and Pont-Lezica, 1977 Daleo etaiy 1977). In one case the product was the a-unsaturated polyprenol phosphate (dehydrodolichol phosphate), which suggests that a-saturation may occur after phosphorylation, at least in tissues such as hen oviduct (Grange and Adair, 1977 Adair and Keller, 1982). 

The initiation step consists in the formation of IPP (12), and its isomer DMAPP (13). The conventional metabolic pathway to form these two molecules is called the acetate/mevalonate pathway (MVA pathway) in which three molecules of acetyl-CoA (3) condense successively to form 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) (5), which leads to a key intermediate molecule, namely mevalonic acid (MVA) (6). The latter is further phosphorylated and decarboxylated to form the IPP molecule (12). In Hevea brasiliensis, this cytosolic pathway was described by Lynen and Lebras" " in the early 1960s and reviewed more recently by Kekwick and Ohya." Most experimental validations were obtained by observing the incorporation of radioactive tracers, such as [2- C] MVA and [3- C]HMG-CoA. The incorporation of [ ClIPP into rubber was found to be much faster than that of [2- C] MVA. This was assumed to be due to slow conversion of MVA into IPP." Another explanation might be that the MVA pathway was not exclusive for IPP biosynthesis. Indeed less than 10 years ago, a new, mevalonate-independent, IPP biosynthesis pathway was discovered by Rohmer." This plastidic DXP-MEP pathway initiates with a... 

The conversion of mevalonate (1) to isopentenyl pyrophosphate (IPP) (4) involves two consecutive phosphorylations at position 5 by successive action of mevalonate kinase (EC 2.7.4.2) and a decarboxylation and dehydration of the tertiary alcohol group by mevalonate 5-pyrophosphate decarboxylase (EC 4.1.1.33) (Fig. 18.4) (Crotean Johnson, 1985 Gershenzon and Croteau, 1990). One mole of ATP is required for each phosphorylation reaction. Mevalonate kinase converts mevalonic acid to (5/ )-phosphomevalonate (5). The second phosphorylation is catalyzed by phospho-mevalonate kinase. The subsequent decarboxylation and dehydration is mediated by the enzyme mevalonate diphosphate decarboxylase (di- or pyrophosphomevalonate decarboxylase EC 4,1.1.3.3) this enzyme requires Mg " or Mn + and ATP for activity (Beale and MacMillan, 1988 Harrison, 1988). All three of these enzymes are found in a number of plants. 

This biosynthetic path consists of the Claisen-type condensation of two acetyl CoA units to form the four-carbon substance acetoacetyl CoA a third equivalent of acetyl CoA is then added in an aldol-type reaction giving, after hydrolysis of one of the thiol esters, hydroxymethylglutaryl CoA (HMG-CoA). HMG-CoA is then reduced by a net four electrons to mevalonic acid and is subsequently phosphorylated to mevalonic acid 5-pyrophosphate (MVA-5PP). This substrate is finally phosphorylated and decarboxylated with concomitant loss of inorganic phosphate to give the five-carbon isoprenoid isopentenyl pyrophosphate (IPP). IPP is then isomerized to DMAPP by an isomerase. 

Enzyme-catalyzed reduction by NADPH (a phosphorylated form of NADH) of the thioester group of 3-hydroxy-3-methylglutatyl-CoA to a primary alcohol gives mevalonic acid, shown here as its anion. 

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