[The Enzymes] Protein Prenylation Part B Volume 30 || The Isoprenoid Biosynthetic Pathway and...

THE ENZYMES,# 2011 Elsevier Inc

12

The Isoprenoid Biosynthetic Pathway

and StatinsSARAH A. HOLSTEIN

Department of Internal Medicine

University of Iowa

Iowa City, Iowa, USA

I. Abstract

The isoprenoid biosynthetic pathway (IBP) is the source of a vast arrayof isoprenoids. This pathway is highly conserved and isoprenoids play keyroles throughout all forms of life. The discovery and development of thestatins, a class of drugs which inhibit the rate-limiting step in the mevalo-nate-dependent IBP, has led to improved scientific understanding of thecomplex regulation of the pathway and has provided therapeutic agentswhich have had far-reaching effects on human health. Here, we provide anoverview of the IBP and discuss the impact of pharmacological manipula-tion of the pathway by the statins.

II. The IsoprenoidBiosynthetic Pathway

A. HISTORICAL OVERVIEW

The IBP (Figure 12.1) is the source of over 23,000 naturally occurringisoprenoids [1]. The first chemical studies of isoprenoids were begun in theearly 1800s (Figure 12.2). Between 1800 and 1884, a large number ofcompounds with the empirical formula of C5H8 were isolated. Otto Wal-lach, in a series of papers published between 1884 and 1887, was the first to

279Vol. XXX ISSN NO: 1874-6047. All rights reserved. DOI: 10.1016/B978-0-12-415922-8.00012-4

Acetyl-CoA

HMG-CoA

Mevalonate

5-Phosphomevalonate

Pyruvate +D-glyceraldehyde3-phosphate

DXPS

1-Deoxy-D-xylulose5-phosphate

IPP

IPP

IPP

GPP

GGPP

Ubiquinone

GeranylgeranylatedProteins

FarnesylatedProteins

Cholesterol

Squalene

FPP

DMAPP

DMAPP

StatinsHMGR

MK

FDPS

FDPS

FTase

GGDPS

GGTases

E-IDS

SQS

FIG. 12.1. The isoprenoid biosynthetic pathway. Intermediates and products are shown in

black and enzymes in blue. Components of the mevalonate-independent DOXP pathway are

shown in green. The HMGR inhibitors (statins) are shown in red.

280 SARAH A. HOLSTEIN

propose what is now known as the ‘‘isoprene rule’’ [2]. He suggested that allterpenes could be built from isoprene (C5H8) units. In the 1950s, LeopoldRuzicka proposed the ‘‘biogenetic isoprene rule’’ which stated that allterpenes could be derived through cyclization or other rearrangementfrom a precursor composed of isoprene units [3]. In 1956, mevalonic acidwas discovered and subsequent studies demonstrated that mevalonic acidcould be incorporated into cholesterol, monoterpenes, rubber, and otherterpenes. By 1957, the pathway for the formation of hydroxymethylglutarylcoenzyme A (HMG-CoA) from acetate was elucidated. Shortly thereafter,the connection between HMG-CoA and mevalonate was made when it wasdemonstrated that HMG-CoA could be enzymatically reduced to

Mevalonic aciddiscovered,found to beincorporatedinto cholesterol

1880s

Wallach’sIsoprene Rule

FPP and GGPPcharacterized

Discovery ofcompactin(mevastatin),the first HMGRinhibitor

Discovery oflovastatin

Discovery of post-translationalmodification ofproteins by a productof mevalonic acid

Brown & Goldsteinwin the Nobel Prize

Identificationof themevalonate-independentDOXPisoprenoidbiosyntheticpathway

1956 1959–1960 1959–1967 1976 1978 1980 1982 1984 1985 1987 1989–1990 1998 2008

HMGR activityidentified as keyenzyme

Brown &Goldsteindemonstratethat treatmentof cells withcompactinresults inupregulation ofHMGR HMGR cloned

FDAapproval oflovastatin

Identification ofprotein farnesylation& geranylgeranylationin higher eukaryotes

Endo receives theLasker Award

FIG. 12.2. Timeline of milestones involving the isoprenoid biosynthetic pathway and statins.


mevalonate. In 1959, Lynen and coworkers reported the characterization ofthe 5-carbon isopentenyl pyrophosphate (IPP) and the 15-carbon farnesylpyrophosphate (FPP) [4]. Soon after, IPP was demonstrated to be anintermediate in the synthesis of squalene [5]. In 1960, Goodman and Popjakidentified dimethylallyl pyrophosphate (DMAPP) and geranyl pyrophos-phate (GPP) as intermediates in the mevalonate-squalene pathway [6].A year later, Grob et al. reported the synthesis of the 20-carbon geranyl-geranyl pyrophosphate (GGPP) from FPP and IPP [7].

B. PRODUCTS OF THE IBP

The IBP and its products are displayed in Figure 12.1. HMG-CoA,ultimately derived from acetyl-CoA is converted to mevalonate via theenzymeHMG-CoA reductase (HMGR) [8]. This reaction is the rate-limitingstep in the pathway. Mevalonate is then phosphorylated via mevalonatekinase (MK) to yield 5-phosphomevalonate [9]. IPP is formed followingadditional phosphorylation and decarboxylation steps [10]. Isomerization ofIPP via the enzyme IPP isomerase yields DMAPP [11]. In mammals, theenzyme farnesyl pyrophosphate synthase (FDPS) catalyzes the synthesis ofbothGPPandFPP [12]. Inplants, a separateGPP synthasehas been identified[13]. GPP is a key intermediate in plants as it serves as the precursor for allmonoterpenes. In animals, however, GPP appears to serve only as an inter-mediate in the synthesis of FPP. Very low basal levels of GPP have beenmeasured in cell culture, although cellular GPP levels can become markedlyincreased in the setting of FDPS inhibition [14].

FPP is necessary for the synthesis of both sterols and longer chainnonsterol isoprenoids. The first committed step in sterol synthesis is cata-lyzed by the enzyme squalene synthesis and involves the head-to-headcondensation of two FPP molecules to form squalene [15]. This is followedby cyclization steps, leading to sterol synthesis. The addition of IPP to FPPvia the enzyme GGPP synthase yields the 20-carbon GGPP [16]. FPP andGGPP are substrates in the prenylation reactions catalyzed by the enzymesfarnesyl transferase (FTase) and geranylgeranyl transferase (GGTase) Iand II [17–20].

Longer chain isoprenoids are synthesized via two other isoprenyl diphos-phate enzyme systems in mammals [21]. Long E-isoprenyl diphosphatesynthase (IDS) produces the side chains of ubiquinone. The length of theside chain varies amongst species, and in humans a C50 synthase has beenidentified [22]. Dehydrodolichyl diphosphate synthase, the only Z-IDSfound in mammals, is responsible for the synthesis of the sugar carriersdolichol and dolichyl phosphate [23]. Plants have additional Z-IDS whichcan catalyze the production of very long isoprene species, such as natural

12. THE ISOPRENOID BIOSYNTHETIC PATHWAY AND STATINS 283

rubber which is composed of over 1000 isoprene units [24]. Most bacteriahave FPP synthase, as well as both E- and Z-long IDS [25–27].

C. THE NONMEVALONATE-DEPENDENT IBP

Plants and bacteria also have a nonmevalonate-dependent IBP, referredto as the deoxy-D-xylulose 5-phosphate (DOXP) pathway (Figure 12.1). Theinitial step in this pathway involves the condensation of pyruvate andD-glyceraldehyde-3-phosphate to form 1-deoxy-D-xylulose 5-phosphate ina reaction catalyzed by deoxyxylulose 5-phosphate synthase (DXPS) [28–30]. Subsequent reactions lead to the synthesis of IPP [31,32]. There iscompartmentalization of isoprenoid biosynthesis in higher plants, such thatthe mevalonate pathway produces sterols, sesquiterpenes, triterpenes, andpolyterpenes in the cytosol while the DOXP pathway synthesizes mono-terpenes, diterpenes, carotenoids, plastoquinones, and the prenyl side chainof chlorophyll in the plastid [33]. In bacteria, the DOXP pathway appears tobe the most ancient pathway and is more common than the mevalonate-dependent pathway [34]. TheDOXPpathwayhas been identified in a varietyof bacteria, mycobacteria, and algae [35–39] but not in fungi or yeasts [40].

III. Statins

A. STATINS AS NATURAL PRODUCTS

In 1976, Endo and coworkers isolated the first HMGR inhibitor, mevas-tatin (compactin) (Figure 12.3), from a culture of Penicillium citrinum [41].During that same year researchers at Beecham Laboratories isolatedmevastatin from Penicillium brevicompactum [42]. Mevastatin was foundto potently inhibit HMGR in vitro with a Ki of 1.4 nM [43], to inhibitcholesterol synthesis in tissue culture cells [44,45], and to reduce plasmacholesterol levels in dogs [46], monkeys [47], and humans [48]. In 1980,lovastatin (mevinolin) was isolated from a strain of Aspergillus terreus [49].HMGR inhibitors have subsequently been isolated from Pleurotus, Mon-ascus, Paecilomyces, Trichoderma, Scopulariopsis, Doratomyces fungalgenera as well as several yeast including Candida cariosilignicola andPichia labacensis [50–53].

Lovastatin and mevastatin are synthesized via polyketide pathways.Polyketides are a large group of structurally diverse secondary metabolitesproduced by bacteria, fungi, and plants. The factors influencing productionof lovastatin or mevastatin have not been fully elucidated. Studies ofAspergillus terreus grown in chemically defined media indicate that

HO

HO

F

F

F

N

OH

HOH OH

OH

OH

N

F

OH

OH

OOH OH

OH

O

O

N

F

N

N

N

SO2Me

OH

OH

OCO2HN N

O

CO2Na

H

Mevastatin Lovastatin

Atorvastatin

PitavastatinCerivastatin

RosuvastatinFluvastatin

Simvastatin Pravastatin

HH3C CH3

O

OO

HO

HHH3C

H3C

CH3

O

OO

HO

HH3C

H3C

CH3CH3

O

OO

HO

HH3C

HO

CH3

H

CO2NaOH

O

FIG. 12.3. Structures of statins.


lovastatin synthesis is initiated after glucose exhaustion and after cessationof lactose consumption, suggesting that lovastatin synthesis occurs in thesetting of starvation conditions [54]. Two genes have been identified thatmay play roles in conferring resistance to compactin. One encodes a proteinwith significant homology to HMGRwhile the other appears to be an effluxpump [55]. A similar mechanism of self-resistance is found in Aspergillusterreus where the lvrA gene encodes a protein related to HMGR [56].

The nature of advantage provided by the production ofHMGR inhibitorsby select fungi is not well understood. Potential explanations include use asinhibitors of environmental competitors or enhancers of their own growth.Althoughmevastatin was initially detected by its antifungal activity [57], themagnitude of this effect was not published. Only 4 out of over 300 strains ofyeast were found to be growth inhibited by compactin analogs [58]. As notedabove, since bacteria predominantly use the mevalonate-independent path-way, it is less likely that theywill be significantly affected by the statins.Therehas been some evidence to suggest that lovastatin can act as an herbicide, at


least with respect to radish seedling growth [59] and cell cultures of Solanumxanthocarpum [60]. Whether this applies more broadly to other plants is notknown. It also is not clear that statins provide a direct growth advantage forthe fungi from which they are derived. Mevinolin production was noted toreach its peak only after the dryweight ofA. terreus had plateaued [53] and isdependent on the composition of the media [60]. Thus the reason for fungalHMGR inhibitor production remains to be determined.

B. SYNTHETIC STATINS

Following the success of lovastatin, a number of other statins weredeveloped (Figure 12.3). Simvastatin, a semisynthetic derivative of lova-statin, was first approved for marketing in Sweden in 1988 and then laterworldwide. Pravastatin, isolated from Nocardia autotropica, was approvedin 1991. The purely synthetic statins fluvastatin (1994), atorvastatin (1997),cerivastatin (1998), rosuvastatin (2003), and pitavastatin (2009) soon fol-lowed. Cerivastatin was subsequently pulled from the market in 2001because of postmarketing surveillance reports which revealed 52 deathsthat were attributed to rhabdomyolysis and resulting renal failure [61].

C. PHARMACOLOGY OF STATINS

All statins share an HMG-like moiety which is linked to rigid hydropho-bic groups (Figure 12.3). Lovastatin and simvastatin are lactone prodrugswhich are converted to the active open hydroxyl acid form in the liver.Enzyme studies show that the statins are competitive inhibitors of HMGRwith respect to HMG-CoA and haveKi values in the 0.1–2.3 nM range [62].Crystal structure studies have revealed that the statins occupy the active sitewhere HMG-CoA binds but do not affect NADPH binding [63].

While statins do inhibit endogenous cholesterol biosynthesis, their hypo-cholesterolemic effect is secondary to increased clearance of LDL from theplasma due to upregulation of the hepatic LDL receptor [64,65]. Althoughthe statins have differing potency, the maximal recommended dose of eachstatin can lead to a similar mean reduction in LDL cholesterol by 35–55%.The majority of the statins are metabolized by the cytochrome P450 system:lovastatin, simvastatin, and atorvastatin are substrates of CYP3A4 whilefluvastatin and rosuvastatin are substrates of CYP2C9 [66]. Pravastatin andpitavastatin, however, are minimally metabolized by the cytochromeP450 system, and therefore have the potential for fewer drug–drug interac-tions [66,67].

There have been several reports suggesting activities of statins unrelatedto HMGR inhibition. Rao et al. reported that the prodrug closed-ring form


of lovastatin inhibited the proteasome [68]. Wojcik et al. also publishedstudies demonstrating the ability of the closed-ring forms of lovastatin andsimvastatin to inhibit proteasome activity, although the authors disagreedwith Rao et al. regarding the effects on the chymotrypsin-like proteaseactivity [69]. These two groups also presented conflicting results with regardto the ability of mevalonate to abrogate the effects of the statin prodrugs onproteasomal activity. Kumar et al. suggested that the open and closed-ringforms of mevastatin differed in their activity as neurotoxic or neuroprotec-tive agents in a cell culture system [70]. Other investigators, however, havereported that these agents do not influence proteasomal activity [71–73]. Atthis time there are no data available to suggest that the putative effect of theclosed-ring form of statins on proteasome activity is clinically meaningful.

D. CLINICAL USE OF STATINS

The statins represent some of the most-widely prescribed drugs in theUnited States and the world. Mevastatin was the first statin to be tested inhumans. In a study involving 11 patients with primary hypercholesterolemia,serumcholesterol levelswere reducedby approximately 30%following dailytreatment for 4–8 weeks [48]. In 1987, lovastatin was the first statin to beapproved for use in humans. Numerous trials have led to various statinsbeing approved for multiple indications, including primary hypercholester-olemia, coronary heart disease, prophylaxis for patients with risk factors forcoronary heart disease, prophylaxis for cerebrovascular accident, hypertri-glyceridemia, and familial hypercholesterolemia. Themajority of the clinicalbenefit of the statins has been attributed to their ability to lower LDL levels.However, there is increasing evidence that statins have pleiotropic effects incardiovascular disease, including effects on endothelial function, atheroscle-rotic plaques, myocardial remodeling and vascular inflammation [74].

There has also been considerable interest in the use of statins in otherclinical indications, including cancer [75], neurological disorders [76], oste-oporosis [77], atrial fibrillation [78], asthma [79], angiogenesis [80], immu-nomodulatory effects [81], coagulation and thrombosis [82,83]. Whetherthese effects can all be attributed to the cholesterol-lowering activity or area consequence of depletion of other isoprenoid species remains to bedetermined.

E. STATINS, MYOPATHY, AND UBIQUINONE

Although statins are generally well tolerated, some patients do developmyopathy. This can range from asymptomatic increases in creatinine kinase(CK) to renal failure from rhabdomyolysis. Risk factors include the dose of


statin, concomitant medications, age, and comorbid conditions [84]. Themechanism of action underlying the statins’ effects on muscle remainsill-defined; however, attention has focused on the role of ubiquinone (coen-zyme Q). Ubiquinone is derived from GGPP (Figure 12.1) and plays animportant role in the electron transport system in mitochondria. Studies inboth animals and humans have demonstrated that statins decrease ubiqui-none blood levels by as much as 50% [85–88]. It has been hypothesized thatstatin-induced decrease in ubiquinone results in mitochondrial dysfunction,causing myotoxicity. Supplementation of ubiquinone was able to restoreplasma levels in patients taking atorvastatin, however this did not correlatewith changes in the CK level [89]. It has also been argued that sinceubiquinone is transported by LDL, that the observed decrease in serum/plasma ubiquinone levels is simply a consequence of the statin-induceddecrease in LDL levels and that tissue levels of ubiquinone may not beaffected [90]. There have been conflicting reports in the literature as towhether tissue ubiquinone levels decrease following statin treatment inboth animal and human studies [87,91–94]. Laaksonen et al. reported anincrease in muscle ubiquinone levels after 1 or 6 months of statin treatmentwhile Paiva et al. reported that simvastatin, but not atorvastatin decreasedmuscle levels [92–94]. Finally, trials evaluating ubiquinone supplementa-tion have yielded equivocal results. Caso et al. reported that supplementa-tion with ubiquinone, but not vitamin E, improved muscle pain symptomswhile Young et al., reported that ubiquinone supplementation did notimprove symptoms [95,96]. Further basic science and clinical studies areneeded to determine both the mechanism of action and management ofstatin-induced myopathy.

F. MK DEFICIENCY

Although a number of genetic disorders associated with isoprenoidbiosynthesis have been identified, the vast majority involve enzymes nec-essary for sterol synthesis. HMGR knock-out mice are embryonic lethal[97]. However, deficiency of MK activity results in two disorders: mevalonicaciduria (MA) and hyper-IgD and periodic fever syndrome (HIDS).Genetic analysis has revealed that the two diseases represent the samedisorder, albeit with differing degrees of severity. MK enzyme activity isundetectable in the fibroblasts of MA patients, while in HIDS patients,activity of 1–7% of control can be found in fibroblasts and leukocytes[98–100]. Despite the nondetectable MK activity in MA fibroblasts, studieshave shown that these cells are capable of synthesizing cholesterol fromradiolabeled acetate [101,102]. In addition, near normal plasma levels ofcholesterol have been found in MA patients [103]. Under control


conditions, levels of prenylated Ras and RhoA proteins in MA and HIDSfibroblasts are similar to control fibroblasts under control conditions [103].However, the patient cells are more sensitive to simvastatin such thataccumulation of cytosolic Ras and RhoA occurs with lower concentrationsof the HMGR inhibitor in the patient cells than in the control cells, consis-tent with the reduced ability of these cells to synthesize FPP and GGPP[103]. As it was hypothesized that excess mevalonate levels were responsi-ble for the clinical features of the disease, two patients with MA wereadministered lovastatin. However, treatment was discontinued followingworsening of their condition [103], suggesting that high mevalonate levelsmay not be the cause of the clinical phenotype and that high HMGRactivity and mevalonate levels are required in order for these cells tomaintain nonsterol synthesis.

IV. Statins and the IBP

A. STATINS AND REGULATION OF THE IBP

Statins have proven to be invaluable tools with which to study theregulation of HMGR, the IBP, and sterol homeostasis. In 1978, Brownand Goldstein used compactin to demonstrate that mevalonate depletionresults in upregulation of HMGR [104]. The generation of a CHO-derivedcell line (UT-1) selected for resistance to compactin and characterized bymarkedly increased levels of HMGR protein, aided in structural studies ofHMGR as well as with the discovery that LDL and 25-hydroxycholesterolaffect HMGR synthesis [105–107]. The roles for both sterol- and nonsterol-mediated regulation of HMGR protein levels were investigated in studiesinvolving statin-treated cells [108–110]. Studies involving the elucidation ofother key regulators of the IBP and sterol homeostasis, including sterolregulatory element-binding proteins (SREBPs), SREBP cleavage-activatingprotein (SCAP), and Insigs, have also incorporated statins [111–114].

B. STATINS AND PRENYLATION

Statins were also instrumental in the discovery of protein prenylation. In1984, Glomset and coworkers used mevinolin (lovastatin) and radiolabeledmevalonate to demonstrate that a product of mevalonate could becomeposttranslationally incorporated into select proteins [115]. The use ofthe statin to deplete cells of endogenous mevalonate and downstreamisoprenoids enabled sufficient incorporation of the exogenous radiolabeledmevalonate and subsequent detection of the radiolabeled protein fraction.


Further studies in the late 1980s revealed that proteins such as lamin Band Ras are farnesylated [116–118]. In the early 1990s, several groupsidentified geranylgeranylated proteins [119–121]. Similar labeling studieswere performed using radiolabeled FPP or GGPP following statin-inducedmevalonate depletion [122]. Innumerable studies have now been publishedutilizing statins to delineate the role of prenylation in modulating proteinfunction.

Statins, by virtue of their ability to deplete cells of all isoprenoid speciesdownstream of mevalonate, including FPP and GGPP, globally diminishprotein prenylation. Submicromolar doses of lovastatin can decrease intra-cellular FPP and GGPP levels in cultured cells [123]; however; under thoseconditions, disruption of protein prenylation is not detected. Work done incultured cells has demonstrated that relative levels of FPP and GGPP varyamongst cell and tissue type, that disparate concentrations of statin areneeded to lower FPP and GGPP levels by equivalent amounts, and thatthere is a hierarchy with respect to the conservation of prenylation ofdifferent prenylated proteins under conditions of mevalonate depletion[123–125] (R.J. Hohl, personal communication). Further studies are neededto better understand the relationship between isoprenoid flux and proteinprenylation.

While a multitude of statin effects have been shown to be due todisruption of protein prenylation in vitro, there is less evidence to suggestthat clinically relevant doses of statins alter protein prenylation in vivo.The concentrations required to limit prenylation in vitro are significantlyhigher (low micromolar) than the concentrations that inhibit cholesterolbiosynthesis (IC50 10 nM) [126]. Therefore, it is generally believed thatunder standard hypercholesterolemia dosing regimens which result inserum drug levels of �0.1 mM [127], cholesterol synthesis is inhibitedbut protein prenylation is conserved. Animal studies utilizing high-dosestatins have shown evidence of disruption of protein prenylation[128,129]. Several phase I studies involving oncology patients have dem-onstrated that administration of high-dose statin can yield serum druglevels in the low micromolar range [130,131]. However, assessment ofprotein prenylation was not described in these reports. In a small studyin which patients with acute myeloid leukemia were given high doses oflovastatin, changes in HMGR activity but not Ras farnesylation weredetected [132]. Thus, whether any of the described pleiotropic effects ofstatins in humans are attributable to disruption of protein prenylation hasyet to be established.

The observation that statins are cytotoxic to a wide variety of cancercells in vitro coupled with the identification of Ras as an importantoncogene, generated much interest in the potential use of statins as


anticancer agents. Phase I and II studies demonstrated that high-dosesof statins were generally well tolerated, although minimal anticanceractivity was noted [130,131,133–135]. As statins have been shown toincrease the cytotoxicity of a wide variety of standard chemotherapeuticagents in vitro, there has also been interest in combination therapy.A number of clinical trials have now been conducted evaluating thecombination of statins and chemotherapy [136–142]. As these trialsinvolve different kinds and doses of statins, multiple different chemo-therapeutic agents, and multiple types of malignancies, it is difficult togenerate a definitive conclusion regarding the efficacy of statins in thissetting. In general however, significant clinical benefit has not beendemonstrated. This may be a consequence of insufficient disruption ofprotein prenylation.

Not only does statin-induced mevalonate depletion affect the functionof prenylated small GTPases, but studies have also revealed an effect onthe expression of the GTPases. It was observed that lovastatin, in addi-tion to diminishing Ras farnesylation in cultured cells, also appeared toincrease the total amount of Ras protein [143]. Subsequent studiesdemonstrated that mevalonate depletion results in the upregulation ofRas and Ras-related proteins by discrete mechanisms including modula-tion of transcriptional, translational, and posttranslational processes[144]. Studies utilizing specific prenyltransferase inhibitors revealedthat inhibition of prenylation was not the signal required for theobserved upregulation but instead was a consequence of depletion ofkey regulatory isoprenoid species [145]. The identification of isoprenoidswith either functional agonist or antagonist properties with respect tothe endogenous isoprenoid pyrophosphates suggested the existence ofspecific isoprenoid-binding factors which are involved in the regulationof Ras-related protein expression [146].

V. FutureDirections

Since their discovery over 30 years ago, statins have proven to beremarkably useful agents in both the basic science and clinical arenas.Worldwide it is estimated that 25 million people are taking these agents.This number could further increase as we learn more about the potentialuse of statins in other disorders. Better understanding of the role of iso-prenoids and isoprenoid-derivatives in human health and disease willundoubtedly lead to the identification of new therapeutic targets andpharmaceutical agents.


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[The Enzymes] Protein Prenylation Part B Volume 30 || The Isoprenoid Biosynthetic Pathway and...

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