IPP-DMAPP isomerase

IPP, and/or DMAPP) can also cause sharp growth inhibition, which can be alleviated by the coexpression of a codon-optimized terpene synthase from the artemisinin pathway [27, 28]. Additionally, the production of lycopene was increased in E. coli by assembling a synthetic mevalonate pathway, which included mevalonate kinase and 5-diphosphomevalonate decarboxylase from yeast, human 5-phosphomevalonate kinase, and E. coli IPP/DMAPP isomerase [29]. Finally, studies on isoprenoid production in S. cerevisiae mainly focused on the mevalonate pathway. For example, Shiba et al. successfully amplified the flux to mevalonate in S. cerevisiae by overexpressing acetyl-CoA synthetase from Salmonella enterica and acetaldehyde dehydrogenase [30]. 

The isomerization of isopentenyl diphosphate to dimethylally diphos phate is catalyzed by JPP isomerase and occurs through a carbocation pathway Protonation of the IPP double bond by a hydrogen-bonded cysteine residue ir the enzyme gives a tertiary carbocation intermediate, which is deprotonated b a glutamate residue as base to yield DMAPP. X-ray structural studies on the enzyme show that it holds the substrate in an unusually deep, well-protectec pocket to shield the highly reactive carbocation from reaction with solvent 01 other external substances. 

The CPPase substrate DMAPP (15) is formed from isopentenyl pyrophosphate (IPP) (14) via the IPP isomerase reaction. It had been assumed that IPP was generated only via mevalonic acid (12) (Fig. 2), but Rohmer discovered another route, 2-C-methyl-D-erythritol 4-phosphate (13) (MEP) pathway (Fig. 2) [22, 23]. A key step in the MEP pathway is the reaction catalyzed by 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which combines hydroxyethyl thiamine pyrophosphate (hydroxyethyl TPP) generated from pyruvic acid (17) and TPP with glyceral-dehyde 3-phosphate (18) to yield 1-deoxy-D-xylulose 5-phosphate (19) containing five carbons. The mevalonate pathway operates in the cytosol of plants and animals, whereas the MEP pathway is present in the plastid of plants or in eubacteria [24-27]. 

Fig. 11 Natural rubber is produced from a side branch of the ubiquitous isoprenoid pathway, with 3-hydroxy-methyl-glutaryl-CoA (HMG-CoA) as the key intermediate derived from acetyl-CoA by the general mevalonic-acid pathway. Mevalonate diphosphate decarboxylase (MPP-D) produces IPP, which is isomeiized to DMAPP by IPP isomerase (IPI). IPP is then condensed in several steps with DMAPP to produce GPP, FPP and GGPP by the action of a trani-prenyltransferase (TPT). The cA-l,4-polymeiization that yields natural rubber is catalyzed by cA-prenyltransferase (CPT), which uses the non-allylic IPP as substrate. Reprinted from [248], with permission from Elsevier...

Recently Fujiwara et al. reported on the in vitro polymerization of trans-polyisoprene using the enzymes isopentenyl diphosphate isomerase (IDI) and fra 3-isoprenyl diphosphate synthase (IDS) [271]. IDI catalyzes the interconversion of IPP and DMAPP. IDS can now catalyze the polymerization of IPP from DMAPP as outlined above for the synthesis of natural rubber, and as outlined in Fig. 13a. However, the condensation process is inhibited due to hydrophobic interaction between IDS and hydrocarbon of the longer products. The hydrophobic chain of the elongating product does not readily protrude into the aqueous phase and it tends to interact with the enzyme. To achieve an efficient in vitro synthesis, the authors used an organic-aqueous two-liquid phase system to successfully synthesize (low molecular weight) fran.y-polyisoprene (see Fig. 13b). 

The IBP and its products are displayed in Figure 12.1. HMG-CoA, ultimately derived from acetyl-CoA is converted to mevalonate via the enzyme HMG-CoA reductase (HMGR) [8]. This reaction is the rate-limiting step in the pathway. Mevalonate is then phosphorylated via mevalonate kinase (MK) to yield 5-phosphomevalonate [9]. IPP is formed following additional phosphorylation and decarboxylation steps [10]. Isomerization of IPP via the enzyme IPP isomerase yields DMAPP [11]. In mammals, the enzyme farnesyl pyrophosphate synthase (FDPS) catalyzes the synthesis of both GPP and FPP [12]. In plants, a separate GPP synthase has been identified [13]. GPP is a key intermediate in plants as it serves as the precursor for all monoterpenes. In animals, however, GPP appears to serve only as an intermediate in the synthesis of FPP. Very low basal levels of GPP have been measured in cell culture, although cellular GPP levels can become markedly increased in the setting of FDPS inhibition [14]. 

Isopentenyl diphosphate (IPP) isomerase catalyzes the conversion of IPP to dimethylallyl diphosphate (DMAPP), an early step in isoprenoid metabolism (Equation (25)). 

DMAPP is a precursor of many isoprenoid compounds including carotenoids, sterols, and ubiquinones. IPP isomerase is an essential enzyme in organisms that use the mevalonate pathway to synthesize isoprenoid units, making the enzymes from S. pneumoniae and S. aureus interesting drug targets. 

The mechanism of type II IPP isomerase is currently not known. Two mechanisms have been proposed. Epoxy and fluorinated substrate analogues have been used to probe the mechanism of type II IPP isomerase. These compounds were irreversible inhibitors of the enzyme forming N5 covalent adducts with the reduced flavin. These studies were taken as evidence of a protonation/deprotonation mechanism in which protonation of the double bond of IPP is followed by deprotonation of the carbocation intermediate to form DMAPP, similar to the mechanism of type I IPP isomerase. Conversely, a radical mechanism has been... 

Isopentenyl diphosphate (IPP) isomerase (EC 5.3.3.2) catalyses the interconversion of IPP and dimethylallyl diphosphate (DMAPP). During a growth cycle of a suspension culture of C. roseus, relatively highest IPP isomerase activity was found between 4 and 6 days after subculture about 20 pkat/mg protein. By Western blot analysis, using purified polyclonal antibodies raised against IPP isomerase from Capsicum annuum chloroplasts, two isoforms of IPP isomerase were detected in the C. roseus extracts [13]. 

Fig. 5.3 Carotenoid biosynthesis in maize endosperm. Compounds IPP, isopentenyl pyrophosphate FPP, famesyl pyrophosphate GGPP, geranylgeranyl pyrophosphate DMAPP, dimethallyl pyrophosphate. Carotenoid biosynthetic pathway enzymes PSY, phytoene synthase PDS, phytoene desaturase ZDS, zetacarotene desaturase ISO, carotene isomerase LCY-B, lycopene beta cyclase LCY-E, lycopene epsilon cyclase HYD-B, beta-carotene hydroxylase HYD-E, alpha-carotene hydroxylase Isonrenoid biosynthetic pathway enzymes IPPI (IPP isomerase) GGPPS (GGPP synthase). Structures are not representative of the geometrical isomer substrates (e.g. Z-phytoene is a bent structure).

After 1,3-allylic isomerization of IPP to dimethylallyl pyrophosphate (DMAPP) by the enzyme IPP isomerase, another IPP unit is added to yield Cio geranylpyro-phosphate (GPP). 

In addition to the formation of IPP (12), initiation includes as well the formation of dimethylallyl diphosphate (DMAPP) (13) by isomerization of IPP, catalyzed by IPP isomerase. 

Tangpakdee also assumed that molecular weight could be regulated by the activity of IPP isomerase, which is necessary to produce DMAPP, and... 

IPP and DMAPP are interconverted by isopentenyl-di-phosphate D-isomerase (isopentenyl pyrophosphate dimethylallyl pyrophosphate isomerase isopentenyl pyrophosphate A -A -isomerase EC 5.3.3.2). This enzyme has been isolated from a number of plant sources, such as pumpkin, orange peel, and pine seedlings. Isopenenyl-diphosphate d-isomerase (from the fungus Claviceps) has a molecular weight of 35,000, a requirement for Mg, and an pH optimum of 6.0-8.5, and it is inhibited by geranyl and famesyl diphosphate. 

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. 

Although, terpenoids show enormous chemical and structural diversity, their backbones are synthesized from only two universal precursors isopentenyl pyrophosphate (IPP) and its highly electrophilic allyUc isomer dimethylaUyl pyrophosphate (DMAPP) [25]. IPP is isomerized to DMAPP by the enzyme isopentenyl pyrophosphate isomerase. The mevalonate pathway for the biosynthesis of terpenoids has been illustrated in Fig. 86.3. To summarize, the active isoprene unit (IPP) is repetitively added to DMAPP or a prenyl diphosphate in sequential head-to-tail... 

A central role in the biosynthesis of isoprenoids is filled by the isopentenyl diphosphate-dimethylallyl diphosphate isomerase (IDl) that catalyzes the interconversion of IPP and DMAPP. The necessity for such an enzyme was suggested in the 1950s when only IPP was known as a monomeric isoprenoid precursor, but an allylic diphosphate such as DMAPP was assumed to have the higher intrinsic reactivity for polyisoprenoid synthesis [22, 88, 89]. The first enzymatic isomerization of IPP to DMAPP was observed in 1959 from a cell-free extract of baker s yeast [90, 91]. Two types of IDI with essentially no amino acid sequence or structural similarities are able to catalyze this interconversion by completely different enzyme mechanisms. The well-known IDI-I have been identified in animals, plants, fungi, and bacteria, whereas the IDI-II can be found mainly in archaea but also in some bacteria [92, 93]. 

As presented in this chapter, today, much is known about the process of terpene biosynthesis. The accumulated knowledge includes a detailed picture about the biosynthesis of the terpenoid monomers IPP and DMAPP either via the mevalonate or the DXP route and their interconversion by isomerases. Also, the stereochemical courses and enzyme mechanisms of all transformations have been largely elucidated. Especially the recently obtained structural data of prenyltransferases and various kinds of terpene synthases resulted in an evolutionary model that involves six domains (a, P, 7,8, e, and Q for the biosynthesis of linear polyisoprenoids from IPP and DMAPP and their subsequent transformation into (poly)cyclic terpenes. All these insights may open up new chances in controlling terpene biosynthesis, e.g., by directed evolution of terpene cyclases or domain swaps in multi-domain enzymes for the production of new terpenes, reconstitution of terpene biosynthetic pathways in heterologous hosts for production optimization, or targeted inhibitirm of pathways in pathogens for disease control. 

The decomposition reaction proceeds in nature by two biosynthetic routes the mevalonate pathway, via mevalonic acid (MVA) (Scheme 102.2), and the recently discovered mevalonate-independent pathway, via deoxyxylulose phosphate (DXP) (Scheme 102.3), the latter taking place in plants. In Scheme 102.2, a schematic representation of the mevalonate pathway is shown in which three molecules of acetyl-coenzyme A derived by carbohydrate, fat, or protein catabolism yield by aldol-type reaction p-hydroxy-(3-methylglutaryl-coenzyme A (HMG-CoA), which is irreversibly reduced to -mevalonic acid (MVA), whose phosphorylation and elimination reaction afford IPP. This molecule is converted by an isomerase into an equilibrium mixture with DMAPP. 

The NR molecule is widely accepted to be synthesized by the successive addition of IPP to dimethylallyl diphosphate (DMAPP) to build up isoprene units in the cis configuration. Initial biochemical studies presumed that IPP, a precursor of rubber molecules from higher plants, is synthesized via the mevalonate (MVA) pathway,which is operated in the cytoplasm from acetyl-Co A. Later, a non-mevalonate pathway called the methylerythritol phosphate (MEP) pathway has also been considered as responsible for IPP synthesis in the plastids from the condensation of pyruvate and glyceraldehyde-3-phos-phate. Mevalonate and l-deoxy-o-xylulose-5-phosphate (DXP) are intermediates in MVA and MEP pathways, respectively. DMAPP is produced by the isomerization of IPP, catalysed by IPP isomerase, from both pathways. The gene expression of DXP synthase in Hevea latex suggested that the MEP pathway for IPP synthesis is involved in rubber biosynthesis.However, an experiment that involved feeding 600 RRIM seedlings with [l- C] 1-deoxy-o-xylulose triacetate, an intermediate derivative of the MEP pathway, indicated that the MEP pathway is involved only in carotenoid biosynthesis but not in the case of rubber biosynthesis. ... 

FIGURE 3.14. Isopentyl diphosphate Isomerase mediates Isomerization of IPP and DMAPP. 

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