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Home , Mevalonate kinase, Mevalonate pathway, Phosphomevalonate kinase

Modified of the archaeon Aeropyrum pernix proceeds via trans- anhydromevalonate 5-phosphate

Hajime 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 upstream MVA pathway exists in almost all and in certain forms biosynthetic route for isoprenoids in general . The partially of , such as bacteria, whereas the vast majority identified pathway has been proposed to explain a mystery of bacteria utilize the methylerythritol phosphate (MEP) pathway surrounding the lack of phosphomevalonate and diphospho- that proceeds through completely different intermediates from mevalonate decarboxylase by the discovery of a conserved , those in the MVA pathway. isopentenyl phosphate kinase. Phosphomevalonate decarboxylase The “modified” MVA pathway was first proposed in 2006 by was considered to be the missing link that would fill the vacancy in Grochowski et al. (4) based on the discovery of a new enzyme, the pathway between mevalonate 5-phosphate and isopentenyl isopentenyl phosphate kinase (IPK), and on data from compar- phosphate. This enzyme was recently discovered from haloarchaea ative analyses of archaeal genomes. For archaea, which do not and certain Chroloflexi bacteria, but their are close homo- possess the MEP pathway, the MVA pathway is requisite for the logs of diphosphomevalonate decarboxylase, which are absent in biosynthesis of specific membrane and other isoprenoids, most archaea. In this study, we used comparative genomic analysis to such as respiratory quinones and . These organisms do Aeropyrum have the putative of most enzymes in the aforementioned

find two enzymes from a hyperthermophilic archaeon, pernix, that can replace phosphomevalonate decarboxylase. One en- -type MVA pathway; it is curious, however, that almost zyme, which has been annotated as putative aconitase, catalyzes the all archaea apparently lack the genes of one or two enzymes of the pathway, typically both PMK and DMD (5–7). Thus, dehydration of mevalonate 5-phosphate to form a previously un- trans Grochowski et al. (4) proposed a bypass pathway, called the known intermediate, -anhydromevalonate 5-phosphate. Then, modified MVA pathway, in which isopentenyl phosphate (IP) another enzyme belonging to the UbiD-decarboxylase family, which was formed from MVA5P by an undiscovered decarboxylase likely requires a UbiX-like partner, converts the intermediate into iso- and was then phosphorylated by IPK, which is conserved in al- pentenyl phosphate. Their activities were confirmed by in vitro assay most all archaea, to yield IPP (Fig. 1A). The decarboxylase [i.e., with recombinant enzymes and were also detected in cell-free extract phosphomevalonate decarboxylase (PMD)] was recently identi- from A. pernix. These data distinguish the modified mevalonate path- fied from a halophilic archaeon, Haloferax volcanii (8), and a way of A. pernix and likely, of the majority of archaea from all known Chloroflexi bacterium, Roseiflexus castenholzii (9). The discovery mevalonate pathways, such as the eukaryote-type classical pathway, substantiated the existence of the proposed modified pathway in the haloarchaea-type modified pathway, and another modified path- these organisms. The pathway is, however, considered to be ex- way recently discovered from Thermoplasma acidophilum. ceptional in the domain Archaea, because the of PMD,

mevalonate pathway | archaea | isoprenoid | dehydratase | decarboxylase Significance

he mevalonate (MVA) pathway provides fundamental pre- Herein, the partially identified “modified” mevalonate path- Tcursors for isoprenoid biosyntheses, such as isopentenyl di- way of the majority of archaea is elucidated using information phosphate (IPP) and dimethylallyl diphosphate (DMAPP). This from comparative genomic analysis. Discovery of two enzymes, pathway was discovered in the late 1950s through the study of mevalonate 5-phosphate dehydratase and trans-anhydromevalonate biosynthesis (Fig. 1A) (1, 2). In this pathway, the C6 5-phosphate decarboxylase, from a hyperthermophilic archaeon, intermediate MVA is formed from acetyl-CoA via acetoacetyl- Aeropyrum pernix, shows that the pathway passes through a CoA and hydroxymethylglutaryl-CoA. It then undergoes two previously unrecognized metabolite, trans-anhydromevalonate steps of phosphorylation catalyzed by (MVK) 5-phosphate. The distribution of the known mevalonate path- and (PMK) to yield mevalonate 5- ways among archaea and other organisms suggests that the A. diphosphate (MVA5PP) via mevalonate 5-phosphate (MVA5P). pernix-type pathway, which is probably conserved among the The C5 compound IPP is synthesized by the of majority of archaea, is the evolutionary prototype for the other MVA5PP accompanied by a detachment of its 3-hydroxyl group. mevalonate pathways involving diphosphomevalonate decar- To catalyze the reaction, diphosphomevalonate decarboxylase boxylase or its homologs. (DMD) consumes ATP to temporarily phosphorylate MVA5PP and form mevalonate 3-phosphate 5-diphosphate inside its cat- Author contributions: T.Y. and H. Hemmi designed research; H. Hayakawa, K.M., F.S., T.I., alytic pocket as shown recently by our mutagenic study (3). H.K., and H. Hemmi performed research; H.K. contributed new reagents/analytic tools; Detachment of the 3-phosphate group of the intermediate triggers H. Hayakawa, F.S., and H. Hemmi analyzed data; and H. Hemmi wrote the paper. decarboxylation to yield IPP. These ATP-dependent enzymes, The authors declare no conflict of interest. MVK, PMK, and DMD, belong to the GHMP (, This article is a PNAS Direct Submission. , mevalonate kinase, phosphomevalonate ki- Published under the PNAS license. nase) kinase family and show a certain level of homology. Con- 1To whom correspondence should be addressed. Email: [email protected]. version of IPP into DMAPP is catalyzed by IPP , which This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. includes two evolutionary independent types of enzymes. This 1073/pnas.1809154115/-/DCSupplemental. most widely accepted, sometimes called “classical” or “canonical,”

www.pnas.org/cgi/doi/10.1073/pnas.1809154115 PNAS Latest Articles | 1of6 Downloaded by guest on October 1, 2021 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 are shown 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 the presence of the putative ortholog genes of described on the left.

which is a close homolog to DMD, is conserved in all haloarchaea This discovery meant that the majority of archaea, in which the but not in most archaea. Different MVA pathways have been found putative orthologs of these enzymes are conserved, likely utilize from other unusual archaea that also possess DMD homologs, such the modified MVA pathway that goes via tAHMP and thus, is as those of the orders Sulfolobales and Thermoplasmatales. The distinct from the known MVA pathways. archaea of the order Sulfolobales, such as Sulfolobus solfataricus, are known to possess a eukaryote-type MVA pathway, but these Results are rare exceptions in archaea (10). In contrast, recent studies Search for Enzymes Involved in the MVA Pathway. To find candi- have proven that the archaea of the order Thermoplasmatales, dates for the undiscovered enzymes involved in the modified such as Thermoplasma acidophilum and Picrophilus torridus,pos- MVA pathway, genes conserved in the archaea that lack the sess a distinctly modified MVA pathway, in which MVA is first genes of DMD homologs were searched from the genomes of 88 converted into mevalonate 3-phosphate (MVA3P) by a DMD archaeal species using the MBGD website (mbgd.genome.ad.jp) homolog, mevalonate 3-kinase (M3K) (Fig. 1A)(11–13). MVA3P that can create sets of putative ortholog genes. The candidate is then phosphorylated by a non-GHMP family kinase, MVA3P 5- genes that we searched for were expected to be absent in the kinase, to form mevalonate 3,5-bisphosphate. The decarboxyl- archaea possessing the DMD homolog genes, such as those of ation of the intermediate is catalyzed by another DMD homolog, the class Halobacteria and the orders Sulfolobales and Ther- bisphosphomevalonate decarboxylase (BMD), to yield IP (14). moplasmatales. By allowing for differences in several genomes, Interestingly, BMD does not require ATP to react, which suggests two gene sets, which are the putative orthologs of A. pernix genes that the two functions of DMD (or PMD), phosphorylation and APE_2087.1 and APE_2089, were selected as the candidates that decarboxylation, were separately inherited by M3K and BMD, best fit the requirements (Fig. 1B and SI Appendix,TableS1). These respectively. Therefore, all of the MVA pathways elucidated to genes of A. pernix likely compose an operon that is annotated in date involve the DMD homologs, which are absent in the great the database as the genes encoding the large and small subunits, majority of archaea (Fig. 1B and SI Appendix,Fig.S1). respectively, of putative aconitase. A group of aconitase homologs This situation motivated us to search for undiscovered en- that includes the A. pernix proteins was previously named “aconitase zymes involved in the MVA pathway of the majority of archaea. X (AcnX)” by Makarova and Koonin (15), and several bacterial We believed that the organisms would possess an isozyme of members of this group were recently shown to catalyze the de- PMD, which shows no homology to DMD. Comparative geno- hydration reactions in hydroxyproline (16, 17). These facts mic analysis, however, led to an unexpected discovery from the suggest the possibility that the proteins APE_2087.1 and APE_2089 are hyperthermophilic archaeon Aeropyrum pernix of two previously the subunits of an enzyme hereafter designated as ApeAcnX, which unidentified enzymes that convert MVA5P into IP via an in- might be a dehydratase or a decarboxylase that catalyzes the de- termediate, trans-anhydromevalonate 5-phosphate (tAHMP). carboxylation evoked by dehydration in the MVA pathway.

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1809154115 Hayakawa et al. Downloaded by guest on October 1, 2021 Identification of MVA5P Dehydratase from A. pernix. Each of the MVA5P or [U-13C]MVA5P also detected ions corresponding to archaeal proteins APE_2087.1 and APE_2089 was recombi- tAHMP (SI Appendix, Figs. S5 and S6). nantly expressed in cells as a fusion with an N- terminal polyhistidine tag. Purification of APE_2087.1 by affinity Identification of tAHMP Decarboxylase. If tAHMP is an in- chromatography yielded a brown-colored solution, which sug- termediate of the MVA pathway of A. pernix, there will be an gested that the has an Fe-S cluster as with other enzyme that connects between tAHMP and IP, because A. aconitase homologs. The protein aggregated immediately af- pernix has a putative ortholog gene of IPK. We noticed that the ter purification, but copurification with APE_2089 yielded stable gene of the UbiD-type decarboxylase homolog likely forms an proteins (Fig. 2A). When copurified with APE_2089, however, operon with the genes of the MVA5P dehydratase subunits in the brown color of APE_2087.1 disappeared in a day after ex- the genomes of some archaea including methanogens, such posure to air. Fig. 2B shows the UV-visible spectrum of the as Methanosarcina acetivorans (SI Appendix,TableS1). Be- APE_2087.1/APE_2089 solution copurified under anaerobic cause UbiD catalyzes the decarboxylation of 3-polyprenyl-4- conditions, the color of which persisted for more than a week. A hydroxybenzoate in the bacterial biosynthetic pathway of ubi- peak at around 400 nm suggests the existence of an Fe-S cluster. quinone (19), this type of decarboxylase is thought to be The solution of the proteins, regarded as ApeAcnX, was reacted involved in the biosynthesis of respiratory quinones that also with radiolabeled intermediates of the MVA pathways, such as are found in some archaea; however, methanogens do not have MVA, MVA5P, and MVA5PP, and the mixtures were analyzed respiratory quinones. This situation implies the involvement of by normal-phase TLC. Only MVA5P was converted into an UbiD-type decarboxylase in the modified MVA pathway of unknown compound with an Rf of 0.50, which is lower than that general archaea. In addition, the above-described candidate of IP at ∼0.6. This showed that ApeAcnX shows enzyme activity genes selected by comparative genomic analysis included, but in other than decarboxylation toward MVA5P (Fig. 2C). Conver- lower ranks, putative ortholog genes encoding UbiD-like pro- sion of MVA and MVA5PP was not observed, which indicates teins and those encoding UbiX-like proteins, which are regar- that the enzyme reaction is highly specific (SI Appendix, Fig. S2). ded as the partners of UbiD-type decarboxylases (20–22) (Fig. The maximum ratio of the product of ApeAcnX to MVA5P was 1B and SI Appendix,TableS1). Although these putative ∼20%, although an excess amount of the enzyme was used for ortholog genes are also found in some archaea utilizing the the reaction, suggesting equilibrium with the substrate. The known MVA pathways, such as several haloarchaea and all product could be recovered from a TLC plate and was reacted archaea of the orders Thermoplasmatales and Sulfolobales, this again with the enzyme (Fig. 2C). After the reaction, a major part might be because their apparent distribution patterns are af-

of the product was converted back into MVA5P. fected by incorporation of the genes of UbiD/UbiX homologs BIOCHEMISTRY To determine the structure of the ApeAcnX product, we responsible for respiratory quinone biosynthesis or other forms performed NMR analysis using a 13C-enriched substrate. The of metabolism. For example, A. pernix has two genes of the enzyme reaction with [U-13C]MVA5P resulted in the emergence putative orthologs of UbiD-type decarboxylase, APE_1571.1 of small NMR signals supposedly derived from the product along and APE_2078; the latter is highly homologous to the UbiD with the signals of unreacted MVA5P (Fig. 2D, Table 1, and SI homolog singly possessed by methanogens and thus, is likely Appendix, Fig. S3). Their chemical shifts and coupling constants involved in the MVA pathway. suggest that the product is derived from the 2,3-dehydration of Thus, we constructed the coexpression system of UbiD and MVA5P. Moreover, the chemical shifts of the emerged signals UbiX homologs from A. pernix (APE_2078 and APE_1647, re- correspond well with those of the trans-anhydromevalonate spectively) in E. coli cells. Because UbiX is known to be a fla- moiety of pestalotiopin A [(E)-5-acetoxy-3-methylpent-2-enoic vin prenyltransferase that produces prenylated flavin mono- acid] (SI Appendix, Fig. S4) reported by Xu et al. (18). These (prFMN), which is a coenzyme required by UbiD facts indicated that ApeAcnX has the activity of MVA5P dehy- (20–22), only APE_2078 was expressed as the fusion protein with dratase, which produces tAHMP. Electrospray ionization–MS a C-terminal polyhistidine tag, while APE_1647 was expressed (ESI-MS) analysis of the reaction products from either nonlabeled without 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-phase TLC 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 ApeAcnX product recovered from TLC and reacted without ApeAcnX; lane 4, the ApeAcnX product recovered from 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 and the ApeAcnX product from [U-13C]MVA5P are in- dicated by overlaying blue and red bars, respectively (SI Appendix,Fig.S3).

Hayakawa et al. PNAS Latest Articles | 3of6 Downloaded by guest on October 1, 2021 Table 1. 13C NMR data for the ApeAcnX product from [U-13C]MVA5P 1 Compound and carbon no. Chemical shift, ppm Coupling pattern* JC-C values, Hz

Product (tAHMP) † 1 177.0 d 260 2 122.8 dd 284/260 3 145.1 ddd (app. td) 284/162/162 4 40.1 dd (app. t) 162/150 5 62.6 d 150

6 (3-CH3) 17.8 d 162 Pestalotiopin A (partial) (18) 1 172.7 —— 2 120.6 —— 3 151.8 —— 440.4—— 563.1——

6 (3-CH3)18.3——

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

purified with a nikkel affinity column (Fig. 3A), we tested its we checked to see if the cell-free extract from A. pernix possessed the enzyme activity by attempting a conversion of radiolabeled enzyme activities that would convert tAHMP into a downstream tAHMP, which had been purified by TLC, into another com- compound, IPP. Radiolabeled putative intermediates of the modified pound. TLC analysis of the reaction mixture showed that a ra- MVA pathway of A. pernix (MVA, MVA5P, tAHMP, IP, and IPP) dioactive spot with an Rf of 0.63 emerged with the disappearance along with an intermediate of the eukaryote-type MVA pathway of the spot of tAHMP (Fig. 3B). Because the Rf value of the (MVA5PP) were reacted with the cell-free extract in the presence of + product approximated that of IP, we verified the formation of IP ATP, Mg2 , S. acidocaldarius GGPP synthase, and DMAPP. Ra- by adding T. acidophilum IPK, Sulfolobus acidocaldarius ger- 2+ diolabeled GGPP was synthesized as the index of IPP formation by anylgeranyl diphosphate (GGPP) synthase, ATP, DMAPP, and Mg the action of enzymes contained in the cell-free extract and was inthesamereaction.Throughthereaction with the enzymes with extracted from the assay mixture with 1-butanol to be analyzed by known functions (23, 24), the product was converted into GGPP as reversed-phase TLC after phosphatase treatment (Fig. 4). The TLC shown by the reversed-phase TLC analysis of the from GGPP autoradiogram indicated that tAHMP could be converted into IPP as in Fig. 3C. This clearly proved that the product was IP, indicating that well as MVA, MVA5P, and IP, whereas the conversion of MVA5PP the UbiD homolog from A. pernix, APE_2078, definitely had tAHMP decarboxylase activity. was not observed. The conversion efficiency of tAHMP was, however, obviously lower than that of the downstream intermediate IP, sug- Verification of the MVA Pathway of A. pernix. Because A. pernix gesting that tAHMP decarboxylase activity in the cell-free extract was possesses the putative genes of the enzymes responsible for the weak. Moreover, the conversion from tAHMP seemed inefficient production of MVA5P from acetyl-CoA and for the conversion even compared with those from the upstream intermediates MVA of IP into downstream metabolites, such as IPP and DMAPP, the and MVA5P. This situation might be explained by the results from discovery of MVA5P dehydratase and tAHMP decarboxylase normal-phase TLC analysis of the assay mixture without GGPP strongly suggests the existence of a modified MVA pathway, synthase and DMAPP (SI Appendix,Fig.S7). IP was completely which passes through the intermediate tAHMP (Fig. 1A). Therefore, converted into IPP by the reaction, showing strong activity of IPK in

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 products from [2-14C]tAHMP. (C) Reversed-phase TLC analysis of 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.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1809154115 Hayakawa et al. Downloaded by guest on October 1, 2021 conceivable evolutionary scenario of the MVA pathways is that PMD emerged first among the DMD homologs, probably via the evolution from some kinase of the GHMP family, and replaced MVA5P dehydratase and tAHMP decarboxylase to create the MVA pathway currently found in haloarchaea and some Chloroflexi bacteria. The replacement caused the additional consumption of an ATP molecule for the production of each molecule of IPP or DMAPP, but it might have allowed the organisms to save a 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 the Aeropyrum-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, and BMD,whichcausedanemergenceoftheeukaryote-typeand the Thermoplasma-type MVA pathways. Based on these ar- guments, we propose that the Aeropyrum-type MVA pathway possessed 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 MVA Fig. 4. Conversion assay with A. pernix cell-free extract. Radiolabeled GGPP ” “ ” was extracted from the reaction mixture containing 14C-labeled intermedi- pathway, and the Thermoplasma-type MVA pathway. ates (A. pernix cell-free extract, ATP, Mg2+, S. acidocaldarius GGPP synthase, and DMAPP) to be analyzed by reversed-phase TLC after phosphatase Materials and Methods

treatment. ori, Origin; s.f., solvent front. Materials. Precoated reversed-phase TLC plates, RP18 F254S, and normal-phase TLC 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 American the cell-free extract. Nevertheless, IP seemed to accumulate in the Radiolabeled Chemicals, Inc. [U-13C]MVA was prepared as described else- reaction with tAHMP, suggesting the inhibition of IPK by tAHMP. where (28). All other chemicals were of analytical grade. BIOCHEMISTRY Discussion Comparative Genomic Analysis. A search for putative ortholog genes dis- In this study, we discovered a modified MVA pathway, which tributed in a certain pattern in representative archaeal species, which were passes through a previously unknown , selected by the one-species-for-each-genus rule, was performed using a web tAHMP, from A. pernix. Unlike the three MVA pathways known to service provided by MBGD (mbgd.genome.ad.jp), allowing some discrepancy date, the fourth MVA pathway lacks the homolog of DMD and (similar pattern search) (29). Multiple alignments of the se- quences of homologous proteins were performed using the online version instead, utilizes two previously unidentified enzymes, MVA5P of the MAFFT program (https://mafft.cbrc.jp/alignment/server/) with default dehydratase and tAHMP decarboxylase. MVA5P dehydratase from settings. Phylogenetic trees were constructed via the neighbor-joining A. pernix is composed of large and small subunits: APE_2087.1 and method using a CLC Sequence Viewer, version 7.5 (CLC bio). APE_2089, respectively. Its putative orthologs from archaea com- prise the type IIb subclass of the AcnX family, while some bac- Enzyme Preparation. Recombinant expression and partial purification of terial AcnX proteins of the types I and IIa subclasses were ApeAcnX (copurified APE_2087.1/APE_2089), APE_2078 (coexpressed with recently revealed to be cis-ortrans-3-hydroxy-L- dehy- APE_1647), R. castenholzii PMD, S. solfataricus MVK, T. acidophilum IPK, and dratase involved in hydroxyproline metabolism (16, 17). This S. acidocaldarius GGPP synthase were performed as described in SI Appen- situation sparked an interest in the evolution of this group of dix, SI Materials and Methods. enzymes along with the unknown function of the remaining type IIc subclass proteins. We also showed that the APE_2078 protein Substrate Preparation. [2-14C]MVA and [2-14C]MVA5PP were prepared from exhibits decarboxylase activity toward tAHMP, which is a unique [2-14C]MVA5P as described elsewhere (10). For the preparation of [4-14C]IP, property for a UbiD homolog, because all known UbiD-type 3.64 nmol [2-14C]MVA5P was reacted with 0.4 mmol purified R. castenholzii μ μ μ decarboxylases react with aromatic substrates, such as 3- PMD, 0.8 mol ATP, 1 mol MgCl2, and 8 mol sodium phosphate, pH 7.5, in polyprenyl-4-hydroxybenzoate and (hydroxy)cinnamic acids (20); a 200-μL reaction mixture. The enzyme was removed by filtration using a the rare exceptions are TtnD from Streptomyces griseochromogenes Vivaspin 500 centrifugation filter (10 kDa molecular weight cut off; GE (25) and SmdK from Streptomyces himastatinicus (26) involved in Healthcare), and the filtrate was used as the solution of [4-14C]IP. the biosynthesis of secondary metabolites. The involvement of the enzyme in the MVA pathway is intriguing, because known UbiD- Radio-TLC Assay of ApeAcnX. To detect the enzyme activity of ApeAcnX, 14 type decarboxylases require prFMN, which is synthesized from a 55 pmol of [2- C]MVA5P was reacted with 17 μgofApeAcnXina30-μLre- probable downstream metabolite of the pathway, dimethylallyl action mixture containing 3 μmol sodium phosphate buffer, pH 8.0. After 1 h μ phosphate. The enzymatic properties of tAHMP decarboxylase, of incubation at 90 °C, a 5- L aliquot of the mixture was spotted on a Silica however, must be thoroughly investigated later. gel 60 normal-phase TLC plate and developed with chloroform/pyridine/ The modified MVA pathway found from A. pernix seems formic acid/water (12:28:6:4). The distribution of radioactivity on the plate was visualized using a Typhoon FLA 9000 imaging analyzer (GE Healthcare) widely distributed among the domain Archaea, with the exceptions and quantified using Image Quant TL software (GE Healthcare). of haloarchaea and the orders Sulfolobales and Thermoplasmatales (Fig. 1B and SI Appendix,Fig.S1). The distribution pattern of the Isolation of the Product of ApeAcnX and Reverse Reaction Assay. A50-μLre- four MVA pathways in the domain Archaea suggests that the action mixture containing 46 nmol [2-14C]MVA5P, 29 μg of ApeAcnX, and modified pathway is more primordial than the other pathways, 5 μmol sodium phosphate buffer, pH 8.0, was incubated at 90 °C for 1 h. All including the eukaryote-type MVA pathway. In contrast, the of the mixture was linearly spotted on a normal TLC plate. After develop- eukaryote-type and haloarchaea-type MVA pathways are pos- ment with the same solvent system used above, the reaction product was

sessed only by a very limited number of species in the domain recovered from the plate by scraping the area around its Rf and washing the Bacteria (SI Appendix,Fig.S8), implying that the pathways in scraped silica gel with 1 M ammonium acetate, pH 7.5. The ammonium ac- bacteria might have horizontal transfer origins. Given the hy- etate solution containing the radiolabeled product was concentrated by pothesis that eukaryotes have evolved from the fusion of archaea heating and used to assay the reverse reaction as [2-14C]tAHMP. and bacteria (27), the modified MVA pathway should be con- For the reverse reaction, a 30-μL reaction mixture containing 55 pmol of sidered the prototype for all known MVA pathways. The most [2-14C]tAHMP, 17 μg of ApeAcnX, and 3 μmol sodium phosphate, pH 8.0, was

Hayakawa et al. PNAS Latest Articles | 5of6 Downloaded by guest on October 1, 2021 incubated at 90 °C for 1 h. TLC analysis was performed as described above pentane. After the addition of 30 nmol farnesol and concentration under an

for the forward reaction. N2 stream, the pentane extract was spotted on an RP-18 F254S reversed-phase TLC plate and developed with an /water (9:1) mixture. The autoradiogram of NMR Analysis. A 300-μL reaction mixture containing 2 μmol [U-13C]MVA, 9 nmol S. the plate was obtained as described above. The same amount of [4-14C]IP was μ μ μ 14 solfataricus MVK, 7.5 mol ATP, 0.3 mol of MgCl2,30 mol sodium phosphate used as a control instead of [2- C]tAHMP in the absence of APE_2078. buffer, pH 7.5, and 10% (vol/vol) D2O was incubated at 60 °C for 3 h. The enzyme μ was removed by filtration using a Vivaspin 500 spin column filter. To 260 Lof Conversion Assay Using Cell-Free Extract from A. pernix. A. pernix was pro- μ the filtrate, 520 g of ApeAcnX was added, and the volume of the solution was vided by the RIKEN BRC through the Natural Bio-Resource Project of the μ adjustedto600 LwithH2OandD2O, keeping the percentage of D2O at 10%. MEXT; cultured at 90 °C in a 250 mL medium, pH 7.0, containing 9.4 g Marine The solution was then incubated at 90 °C for 2 h. After filtration to remove the Broth 2216 (Difco), 1.2 g Hepes-NaOH, and 250 mg Na S O ·5H O; and enzyme, the 13C NMR spectrum of the product from the second reaction was 2 2 3 2 harvested by centrifugation. Then, 0.5 g of the cells were dissolved in 1 mL analyzed using an AVANCE III HD 600 NMR spectrometer equipped with a of 500 mM 3-morpholinopropanesulfonic acid (Mops)-NaOH buffer, pH 7.0, cryoprobe(Bruker).Asanegativecontrol,thesamevolumeofbufferwasadded in place of the ApeAcnX solution. and disrupted by sonication using a Q125 ultrasonic processor (Qsonica). After centrifugation at 22,000 × g for 30 min at 4 °C, the supernatant was MS Analysis. Procedures for negative ion ESI-MS analysis of the products of used as A. pernix cell-free extract. μ MVA5P dehydratase reaction from either nonlabeled MVA or [U-13C]MVA are A100- L reaction mixture containing 0.1 nmol of a radiolabeled substrate 14 14 14 14 14 14 described in SI Appendix, SI Materials and Methods. ([2- C]MVA, [2- C]MVA5P, [2- C]MVA5PP, [2- C]tAHMP, [4- C]IP, or [1- C]IPP), A. pernix cell-free extract containing 200 μg protein, 0.8 μmol ATP, 1 μmol Radio-TLC Assay of APE_2078. A 30-μL reaction mixture containing 55 pmol MgCl2, an excess amount of S. acidocaldarius GGPS, 3 nmol DMAPP, and [2-14C]tAHMP recovered from a TLC plate as described above, 6.2 μg purified Mops-NaOH buffer, pH 7.0, was incubated at 60 °C for 1 h. The radiolabeled APE_2078, and 3 μmol sodium phosphate buffer, pH 7.5, was incubated at GGPP was extracted with 1-butanol and analyzed by reversed-phase TLC 60 °C for 1 h. Normal-phase TLC analysis of the product was performed using after phosphatase treatment as described above. the same procedure described above. Normal-phase TLC analysis of the products from the above reaction To confirm the production of IP, a 100-μL reaction mixture containing without GGPS and DMAPP were performed as described in SI Appendix, SI 14 μ 82 pmol [2- C]tAHMP, 3 g of the purified APE_2078, 0.1 nmol T. acidophilum Materials and Methods. IPK, an excess amount of S. acidocaldarius GGPP synthase, 0.8 μmol ATP, 3 nmol μ DMAPP, 1 mol MgCl2, and sodium phosphate buffer, pH 7.5, was incubated at ACKNOWLEDGMENTS. We thank Kazushi Koga and Atsuo Nakazaki μ 60 °C for 1 h. Then, 200 L of saturated saline was added to the mixture followed (Nagoya University) for help with the NMR analysis. We also thank a μ by the extraction of GGPP with 600 L 1-butanol saturated with saline. Phos- reviewer for suggesting the benefit of the archaeal modified mevalonate phatase treatment of GGPP was performed according to a method described by pathway for anaerobes. This work was partially supported by Grants-in-Aid Fujii et al. (30). To the 1-butanol extract, 2 mL methanol and 1 mL of 0.5 M for Scientific Research (KAKENHI) from JSPS (Japan Society for the Promo- sodium acetate buffer, pH 4.6, containing 6 U acid phosphatase from potato tion of Science) Grants 26660060, 16K14882, and 17H05437 and by grants-in- (Sigma Aldrich) were added. After overnight incubation at 37 °C, geranylger- aid from Takeda Science Foundation, Novozymes Japan, and the Institute for aniol was extracted from the phosphatase reaction mixture with 3 mL n- Fermentation, Osaka (to H. Hemmi).

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