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Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units

Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene Units

Biosci. Biotechnol. Biochem., 66 (8), 1619–1627, 2002

Review Mevalonate and Nonmevalonate Pathways for the of Units

Tomohisa KUZUYAMA

Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan

Isoprenoids are synthesized by consecutive condensa- nate pathway in humans. Its speciˆc inhibitors, tions of their ˆve-carbon , isopentenyl pravastatin and related compounds, are widely used diphosphate, to its isomer, dimethylallyl diphosphate. as -lowering agents.6) Mevalonate is then Two pathways for these precursors are known. One is phosphorylated twice and decarboxylated to form the mevalonate pathway, which operates in eucaryotes, isopentenyl diphosphate (IPP). IPP is then converted archaebacteria, and of higher . The other to its isomer, dimethylallyl diphosphate (DMAPP), is a recently discovered pathway, the nonmevalonate catalyzed by IPP isomerase. IPP and DMAPP syn- pathway, which is used by many eubacteria, green thesized in the mevalonate pathway are used as basic algae, and chloroplasts of higher plants. To date, ˆve units in the biosynthesis of isoprenoids such as reaction steps in this new pathway and their corre- sterols, , and . sponding have been identiˆed. EC numbers of Since the discovery of the mevalonate pathway, it these enzymes have been assigned by the Nomenclature was widely accepted that IPP and DMAPP were Committee of the International Union of formed only through this pathway in all living organ- and Molecular (NC-IUBMB) and are available isms. However, several results inconsistent with the at http:WWwww.chem.qmw.ac.ukWiubmbWenzymeWreac- operation of the mevalonate pathway in certain bac- 13 tionWterpWnonMVA.html. teria had been reported. For example, [ C]acetate, a precursor of the mevalonate pathway, was not incor- Key words: isoprenoid; biosynthesis; mevalonate porated into ubiquinone of 7) or into pathway; nonmevalonate pathway; inhi- pentalenolactone produced by Streptomyces chro- bitor mofuscus (H. Seto, unpublished results).8,9) Feeding experiments with [U-13C] and pentalenolac- In 1956, a newly discovered acetate-replacing tone-producing S. exfoliatus showed labeling pat- factor for Lactobacilli was identiˆed as b-hydroxy- terns inconsistent with the mevalonate pathway.8,9) b-methyl-d-valerolactone by Wolf et al.1) This com- Furthermore, mevinolin, a speciˆc inhibitor of pound also was found to be a precursor of HMG-CoA reductase, did not inhibit the growth of cholesterol.2) In the same year, the chemical structure E. coli.7) These results suggested the existence of an of ``hiochic acid'', a growth factor for Lactobacillus alternative pathway for isoprenoid biosynthesis, homohiochi and L. heterohiochi, also was identiˆed which was not identiˆed for some time. as b-hydroxy-b-methyl-d-valerolactone by Tamura,3) In 1996, Rohmer discovered the ˆrst reaction step whoshowedthatsake (Japanese rice wine) frequently of the alternative, nonmevalonate, pathway for the is spoiled by the that contain this growth formation of IPP and DMAPP in E. coli.10) My factor. Later, b-hydroxy-b-methyl-d-valerolactone group was encouraged by this pioneering work and and ``hiochic acid'' were renamed mevalonate as began to study the nonmevalonate pathway. This their standard name. review centers mostly on our studies of this alterna- In the 1960s, Bloch and Lynen identiˆed the tive pathway. mevalonate pathway for cholesterol biosynthesis.4,5) In this pathway, three molecules of acetyl-CoA 1. First reaction step, catalyzed by DXP condense successively to form 3-hydroxy-3-methyl- synthase (EC 4.1.3.37) glutaryl-CoA (HMG-CoA) (Fig. 1). This CoA- derivative is reduced to mevalonate by HMG-CoA The initial step in the nonmevalonate pathway is reductase, the rate-limiting of the mevalo- the formation of 1-deoxy-D-xylulose 5-phosphate

To whom correspondence should be addressed. Tel: +81-3-5841-7841; Fax: +81-3-5841-8485; E-mail: kuz＠iam.u-tokyo.ac.jp Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopentenyl diphosphate; DMAPP, dimethylallyl diphosphate; DXP, 1-deoxy-D-xylulose 5-phosphate; DX, 1-deoxy-D-xylulose; ME, 2-C-methylerythritol; MEP, 2-C-methyl-D-erythritol 4- phosphate; CDP-ME, 4-(cytidine 5?-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5?-diphospho)-2-C-methyl-D- erythritol; MECDP, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate; HMBDP, 1-hydroxy-2-methyl-2-(E )-butenyl 4-diphosphate 1620 T. KUZUYAMA and a divalent cation such as Mg2+ or Mn2+ for en- zyme activity. The reaction preceding this condensa- tion had earlier been found by Yokota et al.13,14) They detected the formation of 1-deoxy-D-xylulose (DX) from pyruvate and D-glyceraldehyde by pyruvate de- of E. coli or Bacillus subtilis. We cloned a dxs gene homolog from Streptomyces sp. strain CL190 by the polymerase chain reaction using oligonucleotide primers based on highly con- served sequences and compared its en- zymatic properties with those of the E. coli DXP syn- thase.15) Although these two enzymes had diŠerent origins, they had almost identical enzymatic proper- ties. Le áon and co-workers isolated albino mutants of Arabidopsis thaliana. The albino phenotype of one of these mutants was restored by the feeding of DX, the free alcohol of DXP.16) This mutant had a muta- tion in the cla1 gene, which coded for an amino acid sequence very similar to that of E. coli DXP syn- thase. The cla1 gene transcript and its ac- cumulated in young developing tissues. DXP is a biosynthetic intermediate not only for IPP and DMAPP but also for thiamine and pyri- doxol in E. coli (Fig. 2).17) This ˆnding means that DXP synthase is not the only enzyme speciˆc for the nonmevalonate pathway.

2. Second reaction step, catalyzed by DXP reductoisomerase (EC 1.1.1.267)

Rohmer and co-workers found that 2-C- methylerythritol (ME) was incorporated into E. coli cells, which used it as the precursor of the side chain of ubiquinone (Fig. 3(A)).18,19) Because of this ˆnd- ing, the intramolecular rearrangement of DXP had been assumed to yield a hypothetical rearrangement product, 2-C-methylerythrose 4-phosphate, which wasthenconvertedto2-C-methyl-D-erythritol 4- phosphate (MEP) by an unknown reduction process (Fig.3(A)).Forthisreason,itwasassumedthattwo enzymes were involved in the formation of MEP from DXP. Fig. 1. Mevalonate Pathway for the Biosynthesis of IPP and To elucidate the details of this mechanism, we DMAPP. studied the cloning of the gene responsible for MEP In this pathway, three molecules of acetyl-CoA condense suc- cessively to form HMG-CoA. This CoA-derivative is reduced to synthesis. We used the strategy of screening for E. mevalonate by HMG-CoA reductase, the rate-limiting enzyme coli mutants with a metabolic block(s) between DXP of this pathway. Mevalonate is then phosphorylated twice and and MEP. Such mutants would need MEP or ME for decarboxylated to form the isoprene unit, IPP. This unit is then growth. In the screening, we used ME and were able converted to DMAPP, with by IPP isomerase. HMG- to select ten mutants with the desired properties; the CoA, 3-hydroxy-3-methylglutaryl coenzyme A; IPP, isopen- tenyl diphosphate; DMAPP, dimethylallyl diphosphate. addition of ME, but not of DX, to the minimum medium facilitated the growth of the mutants.20) By complementation of these mutants, only the yaeM (DXP) by the condensation of pyruvate and D-glycer- gene, with an unknown function, was cloned.21) All aldehyde 3-phosphate, catalyzed by DXP synthase of the ten mutants that needed ME were conˆrmed to (Fig. 2).10) The dxs gene encoding this enzyme was be yaeM-gene-deˆcient mutants. ˆrst cloned from E. coli.11,12) This enzyme has a typi- Next, to check whether the yaeM gene was in fact cal thiamine-binding motif and needs both thiamine Mevalonate and Nonmevalonate Pathways 1621

Fig. 2. First Step of the Nonmevalonate Pathway. DXP synthase catalyzes the condensation of pyruvate and D-glyceraldehyde 3-phosphate to form DXP, the biosynthetic intermediate not only for IPP and DMAPP but also for thiamine and pydoxol. DX can be incorporated into E. coli and Arabidopsis thaliana to be used as the precursor of these compounds. TPP, thiamine diphosphate; DX, 1-deoxy-D-xylulose.

Fig. 3. Two Reductoisomerase Reactions. (A) DXP reductoisomerase catalyzes simultaneous intramolecular rearrangement and reduction of DXP to form MEP, presumably via the hypothetical reaction intermediate, MEOP. FMM is a speciˆc inhibitor of this enzyme. ME can be incorporated into E. coli to be used as the precursor of IPP and DMAPP. (B) Ketol acid reductoisomerase in valine biosynthesis catalyzes the simultaneous in- tramolecular rearrangement and reduction of 2-acetolactate to form 2,3-dihydroxyisovalerate, presumably via a hypothetical reaction intermediate, 2-oxo-3-hydroxyisovalerate. This hypothetical compound inhibits ketol acid reductoisomerase activity. ME, 2-C- methylerythritol; MEOP, 2-C-methylerythrose 4-phosphate; FMM, fosmidomycin. responsible for MEP formation, we constructed a NADPH level, indicating that the enzyme converted plasmid for the overexpression of the gene product. DXP into an unknown reaction product. We puriˆed The puriˆed enzyme was detected as a homogeneous the reaction product and identiˆed it MEP on the protein band with a subunit size of 42 kDa by sodium basisofNMRandMSdata.20) The result showed that dodecyl sulfate-polyacrylamide gel electrophoresis. MEP is synthesized in the presence of NADPH by The enzyme had a molecular mass of 165 kDa as esti- rearrangement and reduction of DXP in a single step mated by native polyacrylamide gel electrophoresis. (Fig. 3(A)). We designated this enzyme DXP reduc- The enzyme appeared to be a homotetramer. toisomerase and renamed the yaeM gene ``dxr''.21) Incubation of the enzyme with DXP in the The reaction preceding this biosynthetic rearrange- presence of NADPH resulted in a decrease in the ment is involved in the biosynthesis of valine, isoleu- 1622 T. KUZUYAMA

Table 1. Distribution of Genes Involved in the Mevalonate and Nonmevalonate Pathways

Former gene name: yaeM ygbP ychB ygbB gcpE Gene name: hmgr dxs dxr mect cmek mecs

Eubacteria Actinobacillusactinomycetemcomitans ++++++ Aquifexaeolicus ++++++ Bacillussubtilis ++++++ Chlamydiatrachomatis ++++++ Clostridium acetobutylicum + +++++ Deinococcus radiodurans + +++++ Escherichiacoli ++++++ Haemophilus in‰uenzae + +++++ Helicobacterpylori ++++++ Mycobacteriumavium ++++++ Mycobacteriumtuberculosis ++++++ Neisseria gonorrhoeae + +++++ Neisseriameningitidis ++++++ Porphyromonasgingivalis ++++++ Pseudomonasaeruginosa ++++++ Salmonellatyphimurium ++++++ Shewanellaputrefaciens ++++++ Synechocystis sp.strainPCC6803 ++++++ Treponemapallidum ++++++ Yersiniapestis ++++++

Borrelia burgdorferi + Enterococcus faecalis + Staphylococcus aureus + Streptococcus mutans + Streptococcus pneumoniae + Streptococcus pyrogenes +

Archaebacteria Archaeoglobus fulgidus + Methanobacterium thermoautotrophicum + Methanococcus jannaschii +

Eucaryotes Saccharomyces cerevisiae + Arabidopsis thaliana ++* +* +* +* +* +* human +

+showsthegenetobepresent. hmgr,HMG-CoAreductase;dxs, DXP synthase; dxr, DXP reductoisomerase; mect, MEP cytidylyltransferase; cmek,CDP-MEkinase;mecs,MECDP synthase. * These gene products presumably function in , because they have transit peptides in the N-terminal. cine, and . The enzyme in question, ketol acid of the E. coli enzyme is important in the conversion reductoisomerase (EC 1.1.1.86), catalyzes the of DXP to MEP, and His153,His209,andHis257 are rearrangement of 2-acetolactate to yield 2,3-di- part of DXP binding to the enzyme.23) These results hydroxyisovalerate with simultaneous reduction in were supported by those of studies of the three- 22) the presence of NADPH (Fig. 3(B)). It should be dimensional structure of DXP reductoisomerase.24) noted that 2-oxo-3-hydroxy isovalerate, a compound with a keto function, had been excluded as a possible 3. Fosmidomycin, an inhibitor of DXP reaction intermediate because of its inhibitory eŠect reductoisomerase on this reaction.22) As mentioned above, DXP is a precursor not only DXP reductoisomerase is widely distributed in for IPP and DMAPP but also for thiamine and plants and many eubacteria including pathogenic pyridoxol. Therefore, the reaction catalyzed by DXP ones, but not in mammals, so this enzyme could be a reductoisomerase is the committed step in the non- target for screening for herbicides and antibacterial mevalonate pathway. drugs (Table 1).20,21) For rapid screening for DXP We were the ˆrst to identify the catalytic residues reductoisomerase inhibitors, we constructed an assay of DXP reductoisomerase by analysis of the E. coli system that used a microplate reader.25) Before yaeM-gene-deˆcient mutants described above. Glu231 Mevalonate and Nonmevalonate Pathways 1623 screening, we did a literature search to try to ˆnd an- 4. Reaction steps leading to synthesis of tibiotics active against E. coli and B. subtilis,which IPP from MEP both have the nonmevalonate pathway, but inactive against Staphylococcus aureus, which has the How many reaction steps are necessary for the mevalonate pathway; we thought that any inhibitors conversion of MEP into IPP? At least three dehydra- might have already been reported as . Fos- tions and one phosphorylation process would be midomycin (FR-31564) had the expected antibacteri- needed. al spectrum and was identiˆed as a possible DXP To elucidate the later reactions that lead to the syn- reductoisomerase inhibitor. thesis of IPP from MEP, we cloned the genes respon- Fosmidomycin had antibacterial activity against sible for conversion of MEP into IPP. To do this, we most Gram-negative and some Gram-positive bacter- used E. coli mutants with a metabolic block(s) be- ia.26,27) Because it inhibited menaquinone and carote- tween MEP and IPP. Such a mutation would be noid biosynthesis in Micrococcus luteus,itwas lethal, so we engineered an E. coli transformant with proposed that the inhibited the biosynthe- an additional biosynthetic pathway for IPP: the sis of isoprenoids.28) From these studies of fos- mevalonate pathway.30) This transformant with midomycin, we assumed that the antibiotic might in- , , and hibit DXP reductoisomerase. diphosphomevalonate decarboxylase activities can In our assay, fosmidomycin strongly inhibited use part of the mevalonate pathway for IPP biosyn- DXP reductoisomerase in a dose-dependent way with thesis only in the presence of mevalonate. The genes an IC50 of 24 nM. The Lineweaver-Burk plot indicat- encoding these enzymes were cloned from Strep- 30,31) ed competitive inhibition with a Ki value of 9.4 nM. tomyces sp. strain CL190. Mevalonate added to Therefore, fosmidomycin was a competitive inhibitor the growth medium is converted by these three en- of DXP reductoisomerase.25) zymes into IPP, which is then used by the transfor- We next investigated the antibacterial activity of mant as a precursor for isoprenoids. fosmidomycin against E. coli. Bacterial growth was With this transformant as the parent strain, we completely inhibited at 6.25 mgWml FMM. However, obtained mutants with an obligatory requirement of inhibition by the antibiotic of the growth of E. coli mevalonate for growth.32) These mutants had the ex- was suppressed by ME added to the growth medium. pected phenotypes: the addition of mevalonate, but In addition, fosmidomycin had no eŠect on the not ME, to a minimum medium facilitated the growth of an E. coli DXP reductoisomerase dis- growth of the mutants. These phenotypic features ruptant29) inthepresenceofME.Thelackofinhibi- showed unequivocally that the mutants had a tion by fosmidomycin of the disruptant suggested defect(s) in the pathway leading to IPP from MEP. that the disruptant lacked a molecular target. From Using these mutants, we cloned four uncharacterized these results, we concluded that fosmidomycin spe- genes, ygbP, ychB, ygbB, gcpE,thatcomplemented ciˆcally inhibited DXP reductoisomerase in the non- the defects of these blocked mutants in synthesizing mevalonate pathway.25) IPP from MEP (Table 1).32-34) It should be noted that It should be emphasized that fosmidomycin and these four genes apparently are needed for IPP MEOP, the hypothetical rearrangement intermediate biosynthesis in E. coli. The distribution patterns of of DXP reductoisomerase, have close structural the DXP synthase and DXP reductoisomerase genes similarities: they have formyl and phosphonate (or of the nonmevalonate pathway in eubacteria were phosphate) functions separated by ˆve chemical similar (Table 1), supporting the hypothesis that bonds. these four genes are essential for this pathway. As mentioned earlier, the DXP reductoisomerase To identify the functions of the gene products of reaction presumably proceeds in a manner similar to gcpE, ygbB, ychB,andygbP genes, we constructed the ketol acid reductoisomerase reaction. Interesting- plasmids for overexpression of the gene products and ly, 2-oxo-3-hydroxyisovalerate, a compound with a could prepare enough of the enzymes to characterize keto function corresponding to MEOP, inhibits ketol them. Next, we sought to select appropriate reaction acid reductoisomerase,22) which ˆnding supports the conditions for the conversion of MEP into an observation that fosmidomycin inhibits DXP reduc- unknown product by the action of one of the four toisomerase as an analog of MEOP. enzymes. As I mentioned above, we identiˆed the catalytic residues of DXP reductoisomerase23) and clariˆed its 4.1. Third reaction step, catalyzed by MEP crystal structure.24) These results should provide in- cytidylyltransferase (EC 2.7.7.60) formation useful in the development of antibacterial The ygbP gene product converted MEP into an drugs and herbicides. unknown reaction product in the presence of CTP. We puriˆed the reaction product and identiˆed it as 4-(cytidine 5?-diphospho)-2-C-methyl-D-erythritol 1624 T. KUZUYAMA (CDP-ME) on the basis of the results of NMR and MS.32) The results showed that MEP is converted to CDP-ME in the presence of CTP by the ygbP gene product, and that the gene product is essential for IPP biosynthesis (Fig. 4).32) We designated the en- zyme MEP cytidylyltransferase and renamed the ygbP gene ``mect''.32)

4.2. Fourth reaction step, catalyzed by CDP-ME kinase (EC 2.7.1.148) The ychB gene product converted CDP-ME to an unknown reaction product in the presence of ATP. We puriˆed the reaction product and identiˆed it as 2-phospho-4-(cytidine 5?-diphospho)-2-C-methyl-D- erythritol (CDP-ME2P).33) This result showed that CDP-ME is converted to CDP-ME2P in the presence of ATP by the ychB gene product, and that this gene product is essential for IPP biosynthesis (Fig. 4).33) We designated the enzyme 4-(cytidine 5?-diphospho)- 2-C-methyl-D- and renamed the ychB gene ``cmek''.33)

4.3. Fifth reaction step, catalyzed by MECDP synthase (EC 4.6.1.12) The ygbB gene product converted CDP-ME2P to an unknown reaction product. We puriˆed the reaction product and identiˆed it as 2-C-methyl-D- erythritol 2,4-cyclodiphosphate (MECDP).34) The formation of MECDP was concomitant with the elimination of CMP from CDP-ME2P. This result showed that CDP-ME2P is converted to MECDP by the ygbB gene product, and that this gene product is essential for IPP biosynthesis (Fig. 4).34) We designat- ed this enzyme MECDP synthase and renamed the ygbB gene ``mecs''.34) MECDP accumulates in some bacteria, including Corynebacterium ammoniagenes, under oxidative stress caused by benzyl viologen,35) but there have been no reports on the role of MECDP in the non- mevalonate pathway. We showed that MECDP is an intermediate in the nonmevalonate pathway for IPP biosynthesis. Independent of our studies, German researchers Fig. 4. Nonmevalonate Pathway for the Biosynthesis of IPP and found that MECDP is synthesized from MEP by the DMAPP. consecutive actions of the ygbP, ychB,andygbB The initial step of the pathway is the formation of DXP by 36–38) condensation of pyruvate and D-glyceraldehyde 3-phosphate, gene products and that MECDP is e‹ciently catalyzed by DXP synthase. In the second step, DXP is convert- converted into in chromoplasts of Capsi- ed to MEP by DXP reductoisomerase. MEP is then cytidylylat- cum annuum and Narcissus pseudonarcissus.39) ed by MEP cytidylyltransferase to yield CDP-ME, which is phosphorylated by CDP-ME kinase to yield CDP-ME2P. Next, 4.4. Function of the gcpE gene product CDP-ME2P is converted to MECDP as catalyzed by MECDP synthase. IPP and DMAPP could be independently biosynthe- Next, we incubated MECDP with the gene gcpE sized via HMBDP in the nonmevalonate pathway. The subse- product in the presence of various possible cofactors quent reactions leading to the formation of IPP and DMAPP such as NADPH, triphosphates, or from MECDP remain to be elucidated. DXP, 1-deoxy-D-xylu- with the expectation that the gene product lose 5-phosphate; MEP, 2-C-methyl-D-erythritol 4-phosphate; would convert MECDP to an unidentiˆed reaction CDP-ME, 4-(cytidine 5?-diphospho)-2-C-methyl-D-erythritol; CDP-ME2P, 2-phospho-4-(cytidine 5?-diphospho)-2-C-methyl- product. However, attempts to detect this reaction D-erythritol; MECDP, 2-C-methyl-D-erythritol 2,4-cyclo- product in vitro have been unsuccessful. An unidenti- diphosphate; HMBDP, 1-hydroxy-2-methyl-2-(E )-butenyl 4- ˆed gene(s) could be involved in the use of MECDP diphosphate. Mevalonate and Nonmevalonate Pathways 1625 as a . ing for antibacterial drugs, herbicides, and an- timalarial drugs. Screening for nonmevalonate- 5. Recent studies pathway-speciˆc inhibitors is in progress in our laboratory. Recently, studies that used the IPP isomerase- disruptant of E. coli conˆrmed that IPP isomerase is Acknowledgments not essential for growth.40) This ˆnding indicates that IPP and DMAPP are synthesized through indepen- This work is supported by a Grant-in-Aid for dent routes in the late steps of the nonmevalonate Scientiˆc Research on Priority Areas (C) ``Genome pathway. Biology'' from the Ministry of Education, Culture, The lytB gene has a distribution pattern similar to Sports, Science, and Technology of Japan. I thank the patterns of the dxs, dxr, ygbP, ychB, ygbB,and the Japan Society for Bioscience, , and gcpE genes of the nonmevalonate pathway in eubac- Agrochemistry for the Japan Bioscience, Biotechnol- teria, suggesting that the lytB gene is involved in the ogy and Agrochemistry Society Award for the En- nonmevalonate pathway. Although studies of the couragement of Young Scientists. mutated lytB gene supported this suggestion,41) the in vitro function of this gene product has not been eluci- References dated. Very recently, Jomaa et al. reported that the lytB- 1) Wolf,E.D.,HoŠman,H.C.,Aldrich,E.P.,Skeggs, disruptant of E. coli accumulates 1-hydroxy-2- R. H., Wright, D. L., and Folkers, K., b-Hydroxy-b- methyl-2-(E )-butenyl 4-diphosphate (HMBDP).42) methyl-d-valerolactone (divalonic acid), a new bio- logical factor. J. Am. Chem. Soc., 78, 4499 (1956). Arigoni and co-workers demonstrated that a recom- 2) Tavormina, A. P., Gibbs, H. M., and HuŠ, W. J., binant E. coli that overexpresses the xylB,43) dxr, Utilization of b-hydroxy-b-methyl-d-valerolactone in ygbP, ychB, ygbB,andgcpE genes can convert ex- cholesterol biosynthesis. J. Am. Chem. Soc., 78, 44) ogenous DX into HMBDP. In addition, they found 4498–4499 (1956). that a recombinant E. coli overexpressing the xylB, 3) Tamura, G., Hiochic acid, a new growth factor for dxr, ygbP, ychB, ygbB, gcpE,andlytB genes can Lactobacillus homohiochi and Lactobacillus hetero- convert exogenous DX into IPP and DMAPP.45) In hiochi. J. Gen. Appl. Microbiol., 2, 431–434 (1956). these experiments, the xylB gene product catalyzes 4) Katsuki, H., and Bloch, K., Studies on the biosynthe- the phosphorylation of DX to yield DXP in vivo.43) sis of in yeast: Formation of methylated in- These recent ˆndings suggest that MECDP can be termediates. J. Biol. Chem., 242, 222–227 (1967). converted to HMBDP as catalyzed by the gcpE gene 5) Lynen, F., Biosynthetic pathways from acetate to product and that HMBDP can be converted to IPP natural products. Pure Appl. Chem., 14, 137–67 (1967). and DMAPP, catalyzed by the gene product lytB 6) Watanabe, Y., Ito, T., Shiomi, M., Tsujita, Y., (Fig. 4). However, the in vitro reactions catalyzed by Kuroda, M., Arai, M., Fukami, M., and Tamura, A., the gcpE and lytB gene products have not been de- Preventive eŠect of pravastatin sodium, a potent inhi- tected. bitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on coronary atherosclerosis and xanthoma 6. Conclusions in WHHL rabbits. Biochim. Biophys. Acta, 960, 294–302 (1988). The ˆve reactions leading to the synthesis of 7) Zhou, D., and White, R. H., Early steps of isoprenoid MECDP from pyruvate and D-glyceraldehyde 3- biosynthesis in Escherichia coli. Biochem. J., 273, phosphate of the nonmevalonate pathway elucidated 627–634 (1991). 8) Cane, D. E., Rossi, T., and Pachlatko, J. P., The to date are shown in Fig. 4. The genes encoding the biosynthesis of pentalenolactone. Tetrahedron Lett., enzymes that catalyze these ˆve reactions have been 20, 3639–3642 (1979). cloned and characterized. 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