Mevalonate, biosynthesis decarboxylation

D-Mevalonic acid is the fundamental intermediate in the biosynthesis of the terpenoids and steroids, together classed as poly-isoprenoids. The biogenetic isoprene unit is isopentenyl pyrophosphate which arises by enzymic decarboxylation-dehydration of mevalonic acid pyrophosphate. D-Mevalonic acid is almost quantitatively incorporated into cholesterol synthesized by rat liver homogenates. 

Elimination usually involves loss of a proton together with a nucleophilic group such as -OH, -NH3+, phosphate, or pyrophosphate. However, as in Eq. 13-18, step c, electrophilic groups such as -COO-can replace the proton. Another example is the conversion of mevalonic acid-5-pyrophosphate to isopentenyl pyrophosphate (Eq. 13-19) This is a key reaction in the biosynthesis of isoprenoid compounds such as cholesterol and vitamin A (Chapter 22). The phosphate ester formed in step a is a probable intermediate and the reaction probably involves a carbo-cationic intermediate generated by the loss of phosphate prior to the decarboxylation. 

The biosynthesis of steroids begins with the conversion of three molecules of acetyl-CoA into mevalon-ate, the decarboxylation of mevalonate, and its conversion to isopentenyl pyrophosphate. Six molecules of isopentenyl pyrophosphate are polymerized into squalene, which is cyclized to yield lanosterol. Lanos-terol is converted to cholesterol, which is the precursor of bile acids and steroid hormones. 

If sterol content and conformation are so important for membrane stability, we should study the biosynthesis of sterols (Figure 3). The first enzyme in terpenoid biosynthesis is the 3-Hydroxy-3-Methyl-Glutary1-Coenzyme A-reductase (HMG-CoA-reductase) that catalyzes the synthesis of mevalonate. Two phosphorylations and decarboxylation of mevalonate lead to isopentenylpyrophosphate, the basic C -unit in sterol synthesis. Isopentenylpyrophosphate reacts with its isomer, the dimethylally1-pyrophosphate, in a head/tail-reaction to geranyl-pyrophosphate reaction with another C -unit leads to farnesyl-pyro-phosphate, that dimerizes in a tail/tail-reaction to squalene. After expoxidation of its A -double bond, squalene cyclizes to lano-... 

Terpenoids do not necessarily contain exact multiples of five carbons and allowance has to be made for the loss or addition of one or more fragments and possible molecular rearrangements during biosynthesis. In reality the terpenoids are biosynthesized from acetate units derived from the primary metabolism of fatty acids, carbohydrates and some amino acids (see Fig. 2.10). Acetate has been shown to be the sole primary precursor of the terpenoid cholesterol. The major route for terpenoid biosynthesis, the mevalonate pathway, is summarized in Fig. 2.16. Acetyl-CoA is involved in the generation of the C6 mevalonate unit, a process that involves reduction by NADPH. Subsequent decarboxylation during phosphorylation (i.e. addition of phosphate) in the presence of ATP yields the fundamental isoprenoid unit, isopentenyl pyrophosphate (IPP), from which the terpenoids are synthesized by enzymatic condensation reactions. Recently, an alternative pathway has been discovered for the formation of IPP in various eubacteria and plants, which involves the condensation of glyceraldehyde 3-phosphate and pyruvate to form the intermediate 1-deoxy-D-xylulose 5-phosphate (Fig. 2.16 e.g. Eisenreich et al. 1998). We consider some of the more common examples of the main classes of terpenoids below. 

Cholesterol is primarily restricted to eukaryotic cells where it plays a number of roles. Undoubtedly, the most primitive function is as a structural component of membranes. Its metabolism to bile acids and the steroid hormones is relatively recent in the evolutionary sense. In this chapter, the pathway of cholesterol biosynthesis will be divided into segments which correspond to the chemical and biochemical divisions of this biosynthetic route. The initial part of the pathway is the 3-step conversion of acetyl-CoA to 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). The next is the reduction of this molecule to mevalonate, considered to be the rate-controlling step in the biosynthesis of polyisoprenoids. From thence, a series of phosphorylation reactions both activate and decarboxylate mevalonate to isopen tenyl pyrophosphate, the true isoprenoid precursor. After a rearrangement to the allylic pyrophosphate, dimethylallyl pyrophosphate, a sequence of l -4 con-... 

The biosynthesis of the juvenile hormones is not yet fully known. Their similarity to famesol, an intermediate product in the biosynthesis of steroles in mammals, leads one to assume that their formation is analogous to that of farnesol. This obvious assumption seems to be supported by publications that have appeared in the early 1970s. Barnes and Goodfellow (1971) showed that isoprenoid biosynthesis in the larva of Sarcophaga bullata proceeds with the participation of mevalonate kinase. This enzyme regulates the formation of mevalonic acid pyrophosphate, an important intermediate product in steroid biosynthesis of mammals. Isopentenyl pyrophosphate, the C, unit of isoprenoid biosynthesis, is formed from mevalonic acid pyrophosphate by decarboxylation and, with the participation of ATP, by dehydration. 

The ultimate precursor of all the carbon atoms in cholesterol and in the other steroids that are derived from cholesterol is the acetyl group of acetyl-GoA. There are many steps in the biosynthesis of steroids. The condensation of three acetyl groups produces mevalonate, which contains six carbons. Decarboxylation of mevalonate produces the five-carbon isoprene unit frequently encountered in the structure of lipids. The involvement of isoprene imits is a key point in the biosynthesis of steroids and of many other compounds that have the generic name terpenes. Vitamins A, E, and K come from reactions involving terpenes that humans cannot carry out. That is why we must consume these vitamins in our diets vitamin D, the remaining lipid-soluble vitamin, is derived from cholesterol (Section 8.8). Isoprene units are involved in the biosynthesis of ubiquinone (coenzyme Q) and of derivatives of proteins and tRNA with specific five-carbon units attached. Isoprene units are often added to proteins to act as anchors when the protein is attached to a membrane. 

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]. 

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 last step in the biosynthesis of isopentenyl pyrophosphate is the decarboxylation and dehydration of mevalonic pyrophosphate catalyzed by the enzyme mevalonic pyrophosphate anhydrodecarboxylase (E.C. 4.1.1.33). The mechanism by which this reaction occurs is not certain. Lind-berg et al. (1962) proposed a two-step reaction sequence involving phos-... 

The steps required to convert mevalonic acid to the active-isoprenoid intermediate have been worked out with some assurance. The initial step involves the phosphorylation of mevalonic acid to mevalonic acid-5-phosphate by an enzyme called mevalonic kinase. This enzyme was found in yeast by Tchen (1958). The properties of the mevalonic kinase of liver have been described in detail by Levy and PopjAK (1960). The kinase is inhibited by p-chloromercuribenzoate but not by iodoacetamide. The enzyme requires Mg++, Mn++, or Ca++ and ATP or inosine triphosphate. The kinase is specific for the (+) form of mevalonic acid. Mevalonic acid-5-phosphate is phosphorylated further to give mevalonic acid-5-pyrophos-phate (de Waard and Popjak, 1959 Henning et al. 1959). The purified enzyme (Bloch et al., 1959) requires a divalent metal ion for activity (Mg++ is preferable) and has no pronounced pH optimum. Mevalonic acid pyrophosphate then undergoes simultaneous dehydration and decarboxylation to yield isopentenylpyro-phosphate (Lynen et al., 1958 Chaykin et al., 1958). The enzyme concerned with the dehydration and decarboxylation has been purified (Bloch et al., 1959) and shown to have a pH optimum between 5.5 and 7.4 and to require a divalent metal ion (Mg++, Mn++, Fe++ or Co++). The series of reactions in which mevalonate is converted to isopentenylpyrophosphate is outlined in Figure 6. Brodie et al. (1963) have established a new pathway for the biosynthesis of mevalonic acid from malonyl CoA. The importance of this particular pathway in the synthesis of sterols is still unknown. 

The biosynthetic pathway proposed for the synthesis of isopentenyl diphosphate (IPP, isoprene building block) is shown in Figure 4.8 [32]. This pathway combines three acetyl-Co-A molecules to form 3-hydroxy-3-methylglutaryl-Co-A (HMG-CoA). The HMG-CoA is reduced to mevalonic acid which is then phosphorylated and decarboxylated to form IPP, the key building block in terpene biosynthesis synthesis. Lichtenthaler [33] has found that IPP can also be formed via a pathway that does not include mevalonic acid. While there is evidence to support the existence of this alternative pathway, it has not been adequately determined. 

The biosynthesis begins with acetyl CoA which is combined with a second unit of acetyl CoA to give acetoacetyl CoA. A third acetyl CoA molecule is then added and the 6 C body so produced is hydrogenated to mevalonic acid by means of NADPH -I- H+ with the liberation of coenzyme A. Mevalonic acid, which was discovered as a growth factor for microorganisms, is an important intermediate. The active isoprene, iso-pentenyl pyrophosphate, is derived from it by decarboxylation, dehydration, and phosphorylation with ATP. 

Another reaction to isobutene is through decarboxylation and subsequent dehydration of 3-hydroxyisovalerate (3-hydroxy-3-methylbutyrate), catalysed by mevalonate diphosphate decarboxylase (MDD, EC 4.1.1.33) (Gogerty and Bobik 2010 Marliere 2010). This enzyme, from the class of carboxy-lyases, is part of terpenoid or ergosterol biosynthesis, and isobutene formation is its side reaction. Even though the MDD family of enzymes is present in many microorganisms, none of them are known to synthesise isobutene (van Leeuwen et al. 2012 Bloch et al. 1959) (Fig. 4). 

The second step in the biosynthesis of cholesterol is the conversion of mevalonic acid into famesylpyropho-sphate (Fig. 20.19). This is initiated by phosphorylation of the mevalonic acid, followed by decarboxylation yielding isopentylpynrophosphate, which can reversibly isomerise to 3,3-dimethylallylpyrophosphate. 

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