Modified mevalonate pathway of the archaeonAeropyrum pernix proceeds via trans-anhydromevalonate 5-phosphateHajime Hayakawaa, Kento Motoyamaa, Fumiaki Sobuea, Tomokazu Itoa, Hiroshi Kawaideb, Tohru Yoshimuraa,and Hisashi Hemmia,1

aDepartment of Applied Molecular Bioscience, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Aichi, Japan;and bInstitute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, Fuchu, 183-8509 Tokyo, Japan

Edited by C. Dale Poulter, University of Utah, Salt Lake City, UT, and approved August 23, 2018 (received for review May 28, 2018)

The modified mevalonate pathway is believed to be the upstreambiosynthetic route for isoprenoids in general archaea. The partiallyidentified pathway has been proposed to explain a mysterysurrounding the lack of phosphomevalonate kinase and diphospho-mevalonate decarboxylase by the discovery of a conserved enzyme,isopentenyl phosphate kinase. Phosphomevalonate decarboxylasewas considered to be the missing link that would fill the vacancy inthe pathway between mevalonate 5-phosphate and isopentenylphosphate. This enzyme was recently discovered from haloarchaeaand certain Chroloflexi bacteria, but their enzymes are close homo-logs of diphosphomevalonate decarboxylase, which are absent inmost archaea. In this study, we used comparative genomic analysis tofind two enzymes from a hyperthermophilic archaeon, Aeropyrumpernix, that can replace phosphomevalonate decarboxylase. One en-zyme, which has been annotated as putative aconitase, catalyzes thedehydration of mevalonate 5-phosphate to form a previously un-known intermediate, trans-anhydromevalonate 5-phosphate. Then,another enzyme belonging to the UbiD-decarboxylase family, whichlikely requires a UbiX-like partner, converts the intermediate into iso-pentenyl phosphate. Their activities were confirmed by in vitro assaywith recombinant enzymes and were also detected in cell-free extractfrom A. pernix. These data distinguish the modified mevalonate path-way ofA. pernix and likely, of the majority of archaea from all knownmevalonate pathways, such as the eukaryote-type classical pathway,the haloarchaea-type modified pathway, and another modified path-way recently discovered from Thermoplasma acidophilum.

mevalonate pathway | archaea | isoprenoid | dehydratase | decarboxylase

The mevalonate (MVA) pathway provides fundamental pre-cursors for isoprenoid biosyntheses, such as isopentenyl di-phosphate (IPP) and dimethylallyl diphosphate (DMAPP). Thispathway was discovered in the late 1950s through the study ofcholesterol biosynthesis (Fig. 1A) (1, 2). In this pathway, the C6intermediate MVA is formed from acetyl-CoA via acetoacetyl-CoA and hydroxymethylglutaryl-CoA. It then undergoes twosteps of phosphorylation catalyzed by mevalonate kinase (MVK)and phosphomevalonate kinase (PMK) to yield mevalonate 5-diphosphate (MVA5PP) via mevalonate 5-phosphate (MVA5P).The C5 compound IPP is synthesized by the decarboxylation ofMVA5PP accompanied by a detachment of its 3-hydroxyl group.To catalyze the reaction, diphosphomevalonate decarboxylase(DMD) consumes ATP to temporarily phosphorylate MVA5PPand form mevalonate 3-phosphate 5-diphosphate inside its cat-alytic pocket as shown recently by our mutagenic study (3).Detachment of the 3-phosphate group of the intermediate triggersdecarboxylation to yield IPP. These ATP-dependent enzymes,MVK, PMK, and DMD, belong to the GHMP (galactokinase,homoserine kinase, mevalonate kinase, phosphomevalonate ki-nase) kinase family and show a certain level of homology. Con-version of IPP into DMAPP is catalyzed by IPP isomerase, whichincludes two evolutionary independent types of enzymes. Thismost widely accepted, sometimes called “classical” or “canonical,”

MVA pathway exists in almost all eukaryotes and in certain formsof bacteria, such as lactic acid bacteria, whereas the vast majorityof bacteria utilize the methylerythritol phosphate (MEP) pathwaythat proceeds through completely different intermediates fromthose in the MVA pathway.The “modified” MVA pathway was first proposed in 2006 by

Grochowski et al. (4) based on the discovery of a new enzyme,isopentenyl phosphate kinase (IPK), and on data from compar-ative analyses of archaeal genomes. For archaea, which do notpossess the MEP pathway, the MVA pathway is requisite for thebiosynthesis of specific membrane lipids and other isoprenoids,such as respiratory quinones and dolichols. These organisms dohave the putative genes of most enzymes in the aforementionedeukaryote-type MVA pathway; it is curious, however, that almostall archaea apparently lack the genes of one or two enzymesof the pathway, typically both PMK and DMD (5–7). Thus,Grochowski et al. (4) proposed a bypass pathway, called themodified MVA pathway, in which isopentenyl phosphate (IP)was formed from MVA5P by an undiscovered decarboxylaseand was then phosphorylated by IPK, which is conserved in al-most all archaea, to yield IPP (Fig. 1A). The decarboxylase [i.e.,phosphomevalonate decarboxylase (PMD)] was recently identi-fied from a halophilic archaeon, Haloferax volcanii (8), and aChloroflexi bacterium, Roseiflexus castenholzii (9). The discoverysubstantiated the existence of the proposed modified pathway inthese organisms. The pathway is, however, considered to be ex-ceptional in the domain Archaea, because the gene of PMD,

Significance

Herein, the partially identified “modified” mevalonate path-way of the majority of archaea is elucidated using informationfrom comparative genomic analysis. Discovery of two enzymes,mevalonate 5-phosphate dehydratase and trans-anhydromevalonate5-phosphate decarboxylase, from a hyperthermophilic archaeon,Aeropyrum pernix, shows that the pathway passes through apreviously unrecognized metabolite, trans-anhydromevalonate5-phosphate. The distribution of the known mevalonate path-ways among archaea and other organisms suggests that the A.pernix-type pathway, which is probably conserved among themajority of archaea, is the evolutionary prototype for the othermevalonate pathways involving diphosphomevalonate decar-boxylase or its homologs.

Author contributions: T.Y. and H. Hemmi designed research; H. Hayakawa, K.M., F.S., T.I.,H.K., and H. Hemmi performed research; H.K. contributed new reagents/analytic tools;H. Hayakawa, F.S., and H. Hemmi analyzed data; and H. Hemmi wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence should be addressed. Email: hhemmi@agr.nagoya-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809154115/-/DCSupplemental.

Published online September 17, 2018.

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which is a close homolog to DMD, is conserved in all haloarchaeabut not in most archaea. Different MVA pathways have been foundfrom other unusual archaea that also possess DMD homologs, suchas those of the orders Sulfolobales and Thermoplasmatales. Thearchaea of the order Sulfolobales, such as Sulfolobus solfataricus,are known to possess a eukaryote-type MVA pathway, but theseare rare exceptions in archaea (10). In contrast, recent studieshave proven that the archaea of the order Thermoplasmatales,such as Thermoplasma acidophilum and Picrophilus torridus, pos-sess a distinctly modified MVA pathway, in which MVA is firstconverted into mevalonate 3-phosphate (MVA3P) by a DMDhomolog, mevalonate 3-kinase (M3K) (Fig. 1A) (11–13). MVA3Pis then phosphorylated by a non-GHMP family kinase, MVA3P 5-kinase, to form mevalonate 3,5-bisphosphate. The decarboxyl-ation of the intermediate is catalyzed by another DMD homolog,bisphosphomevalonate decarboxylase (BMD), to yield IP (14).Interestingly, BMD does not require ATP to react, which suggeststhat the two functions of DMD (or PMD), phosphorylation anddecarboxylation, were separately inherited by M3K and BMD,respectively. Therefore, all of the MVA pathways elucidated todate involve the DMD homologs, which are absent in the greatmajority of archaea (Fig. 1B and SI Appendix, Fig. S1).This situation motivated us to search for undiscovered en-

zymes involved in the MVA pathway of the majority of archaea.We believed that the organisms would possess an isozyme ofPMD, which shows no homology to DMD. Comparative geno-mic analysis, however, led to an unexpected discovery from thehyperthermophilic archaeon Aeropyrum pernix of two previouslyunidentified enzymes that convert MVA5P into IP via an in-termediate, trans-anhydromevalonate 5-phosphate (tAHMP).

This discovery meant that the majority of archaea, in which theputative orthologs of these enzymes are conserved, likely utilizethe modified MVA pathway that goes via tAHMP and thus, isdistinct from the known MVA pathways.

ResultsSearch for Enzymes Involved in the MVA Pathway. To find candi-dates for the undiscovered enzymes involved in the modifiedMVA pathway, genes conserved in the archaea that lack thegenes of DMD homologs were searched from the genomes of 88archaeal species using the MBGD website (mbgd.genome.ad.jp)that can create sets of putative ortholog genes. The candidategenes that we searched for were expected to be absent in thearchaea possessing the DMD homolog genes, such as those ofthe class Halobacteria and the orders Sulfolobales and Ther-moplasmatales. By allowing for differences in several genomes,two gene sets, which are the putative orthologs of A. pernix genesAPE_2087.1 and APE_2089, were selected as the candidates thatbest fit the requirements (Fig. 1B and SI Appendix, Table S1). Thesegenes of A. pernix likely compose an operon that is annotated inthe database as the genes encoding the large and small subunits,respectively, of putative aconitase. A group of aconitase homologsthat includes the A. pernix proteins was previously named “aconitaseX (AcnX)” by Makarova and Koonin (15), and several bacterialmembers of this group were recently shown to catalyze the de-hydration reactions in hydroxyproline metabolism (16, 17). These factssuggest the possibility that the proteins APE_2087.1 and APE_2089 arethe subunits of an enzyme hereafter designated as ApeAcnX, whichmight be a dehydratase or a decarboxylase that catalyzes the de-carboxylation evoked by dehydration in the MVA pathway.

Fig. 1. Variation and distribution of the MVA pathways. (A) The MVA pathways known to date and discovered in this study. The names of enzymes areshown in boxes, which are colored in light blue, green, or pink when the enzymes are DMD homologs. IDI, isopentenyl diphosphate isomerase. (B) Distri-bution patterns of DMD homologs and the enzymes studied in this work. Each box represents an archaeal species selected on the basis of the one-species-for-each-genus rule (SI Appendix, Table S1). Boxes colored in light blue, green, pink, and gray indicate archaea possessing the (putative) genes of DMD, PMD,M3K/BMD, and a DMD homolog of unknown function, respectively, while white boxes mean their absence. Similarly, boxes colored in red represent thepresence of the putative ortholog genes of proteins described on the left.

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Identification of MVA5P Dehydratase from A. pernix. Each of thearchaeal proteins APE_2087.1 and APE_2089 was recombi-nantly expressed in Escherichia coli cells as a fusion with an N-terminal polyhistidine tag. Purification of APE_2087.1 by affinitychromatography yielded a brown-colored solution, which sug-gested that the protein has an Fe-S cluster as with otheraconitase homologs. The protein aggregated immediately af-ter purification, but copurification with APE_2089 yielded stableproteins (Fig. 2A). When copurified with APE_2089, however,the brown color of APE_2087.1 disappeared in a day after ex-posure to air. Fig. 2B shows the UV-visible spectrum of theAPE_2087.1/APE_2089 solution copurified under anaerobicconditions, the color of which persisted for more than a week. Apeak at around 400 nm suggests the existence of an Fe-S cluster.The solution of the proteins, regarded as ApeAcnX, was reactedwith radiolabeled intermediates of the MVA pathways, such asMVA, MVA5P, and MVA5PP, and the mixtures were analyzedby normal-phase TLC. Only MVA5P was converted into anunknown compound with an Rf of 0.50, which is lower than thatof IP at ∼0.6. This showed that ApeAcnX shows enzyme activityother than decarboxylation toward MVA5P (Fig. 2C). Conver-sion of MVA and MVA5PP was not observed, which indicatesthat the enzyme reaction is highly specific (SI Appendix, Fig. S2).The maximum ratio of the product of ApeAcnX to MVA5P was∼20%, although an excess amount of the enzyme was used forthe reaction, suggesting equilibrium with the substrate. Theproduct could be recovered from a TLC plate and was reactedagain with the enzyme (Fig. 2C). After the reaction, a major partof the product was converted back into MVA5P.To determine the structure of the ApeAcnX product, we

performed NMR analysis using a 13C-enriched substrate. Theenzyme reaction with [U-13C]MVA5P resulted in the emergenceof small NMR signals supposedly derived from the product alongwith the signals of unreacted MVA5P (Fig. 2D, Table 1, and SIAppendix, Fig. S3). Their chemical shifts and coupling constantssuggest that the product is derived from the 2,3-dehydration ofMVA5P. Moreover, the chemical shifts of the emerged signalscorrespond well with those of the trans-anhydromevalonatemoiety of pestalotiopin A [(E)-5-acetoxy-3-methylpent-2-enoicacid] (SI Appendix, Fig. S4) reported by Xu et al. (18). Thesefacts indicated that ApeAcnX has the activity of MVA5P dehy-dratase, which produces tAHMP. Electrospray ionization–MS(ESI-MS) analysis of the reaction products from either nonlabeled

MVA5P or [U-13C]MVA5P also detected ions corresponding totAHMP (SI Appendix, Figs. S5 and S6).

Identification of tAHMP Decarboxylase. If tAHMP is an in-termediate of the MVA pathway of A. pernix, there will be anenzyme that connects between tAHMP and IP, because A.pernix has a putative ortholog gene of IPK. We noticed that thegene of the UbiD-type decarboxylase homolog likely forms anoperon with the genes of the MVA5P dehydratase subunits inthe genomes of some archaea including methanogens, suchas Methanosarcina acetivorans (SI Appendix, Table S1). Be-cause UbiD catalyzes the decarboxylation of 3-polyprenyl-4-hydroxybenzoate in the bacterial biosynthetic pathway of ubi-quinone (19), this type of decarboxylase is thought to beinvolved in the biosynthesis of respiratory quinones that alsoare found in some archaea; however, methanogens do not haverespiratory quinones. This situation implies the involvement ofUbiD-type decarboxylase in the modified MVA pathway ofgeneral archaea. In addition, the above-described candidategenes selected by comparative genomic analysis included, but inlower ranks, putative ortholog genes encoding UbiD-like pro-teins and those encoding UbiX-like proteins, which are regar-ded as the partners of UbiD-type decarboxylases (20–22) (Fig.1B and SI Appendix, Table S1). Although these putativeortholog genes are also found in some archaea utilizing theknown MVA pathways, such as several haloarchaea and allarchaea of the orders Thermoplasmatales and Sulfolobales, thismight be because their apparent distribution patterns are af-fected by incorporation of the genes of UbiD/UbiX homologsresponsible for respiratory quinone biosynthesis or other formsof metabolism. For example, A. pernix has two genes of theputative orthologs of UbiD-type decarboxylase, APE_1571.1and APE_2078; the latter is highly homologous to the UbiDhomolog singly possessed by methanogens and thus, is likelyinvolved in the MVA pathway.Thus, we constructed the coexpression system of UbiD and

UbiX homologs from A. pernix (APE_2078 and APE_1647, re-spectively) in E. coli cells. Because UbiX is known to be a fla-vin prenyltransferase that produces prenylated flavin mono-nucleotide (prFMN), which is a coenzyme required by UbiD(20–22), only APE_2078 was expressed as the fusion protein witha C-terminal polyhistidine tag, while APE_1647 was expressedwithout an affinity tag. Using the APE_2078 protein partially

Fig. 2. Elucidation of the function of ApeAcnX. (A)SDS/PAGE of copurified ApeAcnX. (B) UV-visible spec-trum of 4 mg/mL ApeAcnX solution. (C) Normal-phaseTLC analysis of the ApeAcnX reaction product. Lane 1,[2-14C]MVA5P reacted without ApeAcnX; lane 2, [2-14C]MVA5P reacted with ApeAcnX; lane 3, the ApeAcnXproduct recovered from TLC and reacted withoutApeAcnX; lane 4, the ApeAcnX product recoveredfrom TLC and reacted with ApeAcnX. ori, Origin; s.f.,solvent front. (D) 13C-NMR spectra of the samples be-fore (Left) and after (Right) reaction with ApeAcnX.Signals derived from the substrate [U-13C]MVA5P andthe ApeAcnX product from [U-13C]MVA5P are in-dicated by overlaying blue and red bars, respectively (SIAppendix, Fig. S3).

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purified with a nikkel affinity column (Fig. 3A), we tested itsenzyme activity by attempting a conversion of radiolabeledtAHMP, which had been purified by TLC, into another com-pound. TLC analysis of the reaction mixture showed that a ra-dioactive spot with an Rf of 0.63 emerged with the disappearanceof the spot of tAHMP (Fig. 3B). Because the Rf value of theproduct approximated that of IP, we verified the formation of IPby adding T. acidophilum IPK, Sulfolobus acidocaldarius ger-anylgeranyl diphosphate (GGPP) synthase, ATP, DMAPP, andMg2+

in the same reaction. Through the reaction with the enzymes withknown functions (23, 24), the product was converted into GGPP asshown by the reversed-phase TLC analysis of the alcohol fromGGPPin Fig. 3C. This clearly proved that the product was IP, indicating thatthe UbiD homolog fromA. pernix, APE_2078, definitely had tAHMPdecarboxylase activity.

Verification of the MVA Pathway of A. pernix. Because A. pernixpossesses the putative genes of the enzymes responsible for theproduction of MVA5P from acetyl-CoA and for the conversionof IP into downstream metabolites, such as IPP and DMAPP, thediscovery of MVA5P dehydratase and tAHMP decarboxylasestrongly suggests the existence of a modified MVA pathway,which passes through the intermediate tAHMP (Fig. 1A). Therefore,

we checked to see if the cell-free extract from A. pernix possessed theenzyme activities that would convert tAHMP into a downstreamcompound, IPP. Radiolabeled putative intermediates of the modifiedMVA pathway of A. pernix (MVA, MVA5P, tAHMP, IP, and IPP)along with an intermediate of the eukaryote-type MVA pathway(MVA5PP) were reacted with the cell-free extract in the presence ofATP, Mg2+, S. acidocaldarius GGPP synthase, and DMAPP. Ra-diolabeled GGPP was synthesized as the index of IPP formation bythe action of enzymes contained in the cell-free extract and wasextracted from the assay mixture with 1-butanol to be analyzed byreversed-phase TLC after phosphatase treatment (Fig. 4). The TLCautoradiogram indicated that tAHMP could be converted into IPP aswell as MVA, MVA5P, and IP, whereas the conversion of MVA5PPwas not observed. The conversion efficiency of tAHMPwas, however,obviously lower than that of the downstream intermediate IP, sug-gesting that tAHMP decarboxylase activity in the cell-free extract wasweak. Moreover, the conversion from tAHMP seemed inefficienteven compared with those from the upstream intermediates MVAand MVA5P. This situation might be explained by the results fromnormal-phase TLC analysis of the assay mixture without GGPPsynthase and DMAPP (SI Appendix, Fig. S7). IP was completelyconverted into IPP by the reaction, showing strong activity of IPK in

Table 1. 13C NMR data for the ApeAcnX product from [U-13C]MVA5P

Compound and carbon no. Chemical shift, ppm Coupling pattern* 1JC-C values, Hz

Product (tAHMP)1 177.0 d† 2602 122.8 dd 284/2603 145.1 ddd (app. td) 284/162/1624 40.1 dd (app. t) 162/1505 62.6 d 1506 (3-CH3) 17.8 d 162

Pestalotiopin A (partial) (18)1 172.7 — —2 120.6 — —3 151.8 — —4 40.4 — —5 63.1 — —6 (3-CH3) 18.3 — —

app., Apparent; d, doublet; t, triplet.*Patterns resulted from 1JC-C coupling are indicated.†An additional 25-Hz coupling, which might have resulted from 3JC-C coupling with C4, was observed, whereas acorresponding coupling was not clearly observed with the relatively broad signal of C4. The coupling mightcontribute to the broadening of the C4 signal along with the 3JC-P coupling.

Fig. 3. Elucidation of the function of APE_2078. (A)SDS/PAGE of a partially purified APE_2078. (B)Normal-phase TLC analysis of the reaction productsfrom [2-14C]tAHMP. (C) Reversed-phase TLC analysisof the hydrolyzed products from the reaction with[2-14C]tAHMP or [4-14C]IP in the presence of T.acidophilum IPK and S. acidocaldarius GGPP syn-thase. ori, Origin; s.f., solvent front.

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the cell-free extract. Nevertheless, IP seemed to accumulate in thereaction with tAHMP, suggesting the inhibition of IPK by tAHMP.

DiscussionIn this study, we discovered a modified MVA pathway, whichpasses through a previously unknown metabolic intermediate,tAHMP, from A. pernix. Unlike the three MVA pathways known todate, the fourth MVA pathway lacks the homolog of DMD andinstead, utilizes two previously unidentified enzymes, MVA5Pdehydratase and tAHMP decarboxylase. MVA5P dehydratase fromA. pernix is composed of large and small subunits: APE_2087.1 andAPE_2089, respectively. Its putative orthologs from archaea com-prise the type IIb subclass of the AcnX family, while some bac-terial AcnX proteins of the types I and IIa subclasses wererecently revealed to be cis- or trans-3-hydroxy-L-proline dehy-dratase involved in hydroxyproline metabolism (16, 17). Thissituation sparked an interest in the evolution of this group ofenzymes along with the unknown function of the remaining typeIIc subclass proteins. We also showed that the APE_2078 proteinexhibits decarboxylase activity toward tAHMP, which is a uniqueproperty for a UbiD homolog, because all known UbiD-typedecarboxylases react with aromatic substrates, such as 3-polyprenyl-4-hydroxybenzoate and (hydroxy)cinnamic acids (20);the rare exceptions are TtnD from Streptomyces griseochromogenes(25) and SmdK from Streptomyces himastatinicus (26) involved inthe biosynthesis of secondary metabolites. The involvement of theenzyme in the MVA pathway is intriguing, because known UbiD-type decarboxylases require prFMN, which is synthesized from aprobable downstream metabolite of the pathway, dimethylallylphosphate. The enzymatic properties of tAHMP decarboxylase,however, must be thoroughly investigated later.The modified MVA pathway found from A. pernix seems

widely distributed among the domain Archaea, with the exceptionsof haloarchaea and the orders Sulfolobales and Thermoplasmatales(Fig. 1B and SI Appendix, Fig. S1). The distribution pattern of thefour MVA pathways in the domain Archaea suggests that themodified pathway is more primordial than the other pathways,including the eukaryote-type MVA pathway. In contrast, theeukaryote-type and haloarchaea-type MVA pathways are pos-sessed only by a very limited number of species in the domainBacteria (SI Appendix, Fig. S8), implying that the pathways inbacteria might have horizontal transfer origins. Given the hy-pothesis that eukaryotes have evolved from the fusion of archaeaand bacteria (27), the modified MVA pathway should be con-sidered the prototype for all known MVA pathways. The most

conceivable evolutionary scenario of the MVA pathways is thatPMD emerged first among the DMD homologs, probably via theevolution from some kinase of the GHMP family, and replacedMVA5P dehydratase and tAHMP decarboxylase to create theMVA pathway currently found in haloarchaea and some Chloroflexibacteria. The replacement caused the additional consumptionof an ATP molecule for the production of each molecule of IPPor DMAPP, but it might have allowed the organisms to savea portion of the cost for producing multiple proteins and a spe-cific coenzyme or to avoid the use of an oxygen-sensitive enzyme.PMD seems suitable for aerobes, such as haloarchaea, while theAeropyrum-type modified MVA pathway with lower ATP re-quirement can benefit anaerobes, in which ATP is in short supply.PMD evolved later into other homologs, such as DMD, M3K, andBMD, which caused an emergence of the eukaryote-type andthe Thermoplasma-type MVA pathways. Based on these ar-guments, we propose that the Aeropyrum-type MVA pathwaypossessed by the majority of archaea should be called the “ar-chaeal MVA pathway,” while the others could be called the“(eukaryotic) MVA pathway,” the “haloarchaea-type MVApathway,” and the “Thermoplasma-type MVA pathway.”

Materials and MethodsMaterials. Precoated reversed-phase TLC plates, RP18 F254S, and normal-phaseTLC plates, Silica gel 60, were purchased from Merck Millipore. [2-14C]MVA5P (55 Ci/mol) and [1-14C]IPP (55 Ci/mol) were purchased from AmericanRadiolabeled Chemicals, Inc. [U-13C]MVA was prepared as described else-where (28). All other chemicals were of analytical grade.

Comparative Genomic Analysis. A search for putative ortholog genes dis-tributed in a certain pattern in representative archaeal species, which wereselected by the one-species-for-each-genus rule, was performed using a webservice provided by MBGD (mbgd.genome.ad.jp), allowing some discrepancy(similar pattern search) (29). Multiple alignments of the amino acid se-quences of homologous proteins were performed using the online versionof the MAFFT program (https://mafft.cbrc.jp/alignment/server/) with defaultsettings. Phylogenetic trees were constructed via the neighbor-joiningmethod using a CLC Sequence Viewer, version 7.5 (CLC bio).

Enzyme Preparation. Recombinant expression and partial purification ofApeAcnX (copurified APE_2087.1/APE_2089), APE_2078 (coexpressed withAPE_1647), R. castenholzii PMD, S. solfataricus MVK, T. acidophilum IPK, andS. acidocaldarius GGPP synthase were performed as described in SI Appen-dix, SI Materials and Methods.

Substrate Preparation. [2-14C]MVA and [2-14C]MVA5PP were prepared from[2-14C]MVA5P as described elsewhere (10). For the preparation of [4-14C]IP,3.64 nmol [2-14C]MVA5P was reacted with 0.4 mmol purified R. castenholziiPMD, 0.8 μmol ATP, 1 μmol MgCl2, and 8 μmol sodium phosphate, pH 7.5, ina 200-μL reaction mixture. The enzyme was removed by filtration using aVivaspin 500 centrifugation filter (10 kDa molecular weight cut off; GEHealthcare), and the filtrate was used as the solution of [4-14C]IP.

Radio-TLC Assay of ApeAcnX. To detect the enzyme activity of ApeAcnX,55 pmol of [2-14C]MVA5P was reacted with 17 μg of ApeAcnX in a 30-μL re-action mixture containing 3 μmol sodium phosphate buffer, pH 8.0. After 1 hof incubation at 90 °C, a 5-μL aliquot of the mixture was spotted on a Silicagel 60 normal-phase TLC plate and developed with chloroform/pyridine/formic acid/water (12:28:6:4). The distribution of radioactivity on the platewas visualized using a Typhoon FLA 9000 imaging analyzer (GE Healthcare)and quantified using Image Quant TL software (GE Healthcare).

Isolation of the Product of ApeAcnX and Reverse Reaction Assay. A 50-μL re-action mixture containing 46 nmol [2-14C]MVA5P, 29 μg of ApeAcnX, and5 μmol sodium phosphate buffer, pH 8.0, was incubated at 90 °C for 1 h. Allof the mixture was linearly spotted on a normal TLC plate. After develop-ment with the same solvent system used above, the reaction product wasrecovered from the plate by scraping the area around its Rf and washing thescraped silica gel with 1 M ammonium acetate, pH 7.5. The ammonium ac-etate solution containing the radiolabeled product was concentrated byheating and used to assay the reverse reaction as [2-14C]tAHMP.

For the reverse reaction, a 30-μL reaction mixture containing 55 pmol of[2-14C]tAHMP, 17 μg of ApeAcnX, and 3 μmol sodium phosphate, pH 8.0, was

Fig. 4. Conversion assay with A. pernix cell-free extract. Radiolabeled GGPPwas extracted from the reaction mixture containing 14C-labeled intermedi-ates (A. pernix cell-free extract, ATP, Mg2+, S. acidocaldarius GGPP synthase,and DMAPP) to be analyzed by reversed-phase TLC after phosphatasetreatment. ori, Origin; s.f., solvent front.

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incubated at 90 °C for 1 h. TLC analysis was performed as described abovefor the forward reaction.

NMR Analysis.A 300-μL reaction mixture containing 2 μmol [U-13C]MVA, 9 nmol S.solfataricus MVK, 7.5 μmol ATP, 0.3 μmol of MgCl2, 30 μmol sodium phosphatebuffer, pH 7.5, and 10% (vol/vol) D2Owas incubated at 60 °C for 3 h. The enzymewas removed by filtration using a Vivaspin 500 spin column filter. To 260 μL ofthe filtrate, 520 μg of ApeAcnX was added, and the volume of the solution wasadjusted to 600 μL with H2O and D2O, keeping the percentage of D2O at 10%.The solution was then incubated at 90 °C for 2 h. After filtration to remove theenzyme, the 13C NMR spectrum of the product from the second reaction wasanalyzed using an AVANCE III HD 600 NMR spectrometer equipped with acryoprobe (Bruker). As a negative control, the same volume of buffer was addedin place of the ApeAcnX solution.

MS Analysis. Procedures for negative ion ESI-MS analysis of the products ofMVA5P dehydratase reaction from either nonlabeledMVA or [U-13C]MVA aredescribed in SI Appendix, SI Materials and Methods.

Radio-TLC Assay of APE_2078. A 30-μL reaction mixture containing 55 pmol[2-14C]tAHMP recovered from a TLC plate as described above, 6.2 μg purifiedAPE_2078, and 3 μmol sodium phosphate buffer, pH 7.5, was incubated at60 °C for 1 h. Normal-phase TLC analysis of the product was performed usingthe same procedure described above.

To confirm the production of IP, a 100-μL reaction mixture containing82 pmol [2-14C]tAHMP, 3 μg of the purified APE_2078, 0.1 nmol T. acidophilumIPK, an excess amount of S. acidocaldarius GGPP synthase, 0.8 μmol ATP, 3 nmolDMAPP, 1 μmol MgCl2, and sodium phosphate buffer, pH 7.5, was incubated at60 °C for 1 h. Then, 200 μL of saturated saline was added to themixture followedby the extraction of GGPP with 600 μL 1-butanol saturated with saline. Phos-phatase treatment of GGPP was performed according to a method described byFujii et al. (30). To the 1-butanol extract, 2 mL methanol and 1 mL of 0.5 Msodium acetate buffer, pH 4.6, containing 6 U acid phosphatase from potato(Sigma Aldrich) were added. After overnight incubation at 37 °C, geranylger-aniol was extracted from the phosphatase reaction mixture with 3 mL n-

pentane. After the addition of 30 nmol farnesol and concentration under anN2 stream, the pentane extract was spotted on an RP-18 F254S reversed-phase TLCplate and developed with an acetone/water (9:1) mixture. The autoradiogram ofthe plate was obtained as described above. The same amount of [4-14C]IP wasused as a control instead of [2-14C]tAHMP in the absence of APE_2078.

Conversion Assay Using Cell-Free Extract from A. pernix. A. pernix was pro-vided by the RIKEN BRC through the Natural Bio-Resource Project of theMEXT; cultured at 90 °C in a 250 mL medium, pH 7.0, containing 9.4 g MarineBroth 2216 (Difco), 1.2 g Hepes-NaOH, and 250 mg Na2S2O3·5H2O; andharvested by centrifugation. Then, 0.5 g of the cells were dissolved in 1 mLof 500 mM 3-morpholinopropanesulfonic acid (Mops)-NaOH buffer, pH 7.0,and disrupted by sonication using a Q125 ultrasonic processor (Qsonica).After centrifugation at 22,000 × g for 30 min at 4 °C, the supernatant wasused as A. pernix cell-free extract.

A 100-μL reaction mixture containing 0.1 nmol of a radiolabeled substrate([2-14C]MVA, [2-14C]MVA5P, [2-14C]MVA5PP, [2-14C]tAHMP, [4-14C]IP, or [1-14C]IPP),A. pernix cell-free extract containing 200 μg protein, 0.8 μmol ATP, 1 μmolMgCl2, an excess amount of S. acidocaldarius GGPS, 3 nmol DMAPP, andMops-NaOH buffer, pH 7.0, was incubated at 60 °C for 1 h. The radiolabeledGGPP was extracted with 1-butanol and analyzed by reversed-phase TLCafter phosphatase treatment as described above.

Normal-phase TLC analysis of the products from the above reactionwithout GGPS and DMAPP were performed as described in SI Appendix, SIMaterials and Methods.

ACKNOWLEDGMENTS. We thank Kazushi Koga and Atsuo Nakazaki(Nagoya University) for help with the NMR analysis. We also thank areviewer for suggesting the benefit of the archaeal modified mevalonatepathway for anaerobes. This work was partially supported by Grants-in-Aidfor Scientific Research (KAKENHI) from JSPS (Japan Society for the Promo-tion of Science) Grants 26660060, 16K14882, and 17H05437 and by grants-in-aid from Takeda Science Foundation, Novozymes Japan, and the Institute forFermentation, Osaka (to H. Hemmi).

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