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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: [email protected].
This article contains supporting information online at
www.pnas.org/lookup/suppl/doi:10.1073/pnas.1809154115/-/DCSupplemental.
Published online September 17, 2018.
10034–10039 | PNAS | October 2, 2018 | vol. 115 | no. 40
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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.
10038 | www.pnas.org/cgi/doi/10.1073/pnas.1809154115 Hayakawa et
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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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