Biosci. Biotechnol. Biochem., 66 (8), 1619–1627, 2002 Review Mevalonate and Nonmevalonate Pathways for the Biosynthesis of Isoprene 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 precursor, isopentenyl pravastatin and related compounds, are widely used diphosphate, to its isomer, dimethylallyl diphosphate. as cholesterol-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 cytosols of higher plants. 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, carotenoids, and dolichols. sponding enzymes 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 Biochemistry formed only through this pathway in all living organ- and Molecular Biology (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 Escherichia coli 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or into pathway; nonmevalonate pathway; inhi- pentalenolactone produced by Streptomyces chro- bitor mofuscus (H. Seto, unpublished results).8,9) Feeding experiments with [U-13C]glucose and pentalenolac- In 1956, a newly discovered acetate-replacing tone-producing S. exfoliatus showed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 bacteria 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 enzyme 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- hydrogenase 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 amino acid 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 protein 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 catalysis 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.