Biosynthesis of Ether-Type Polar Lipids in Archaea and Evolutionary

24 Pages·2007·1.05 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,Mar.2007,p.97–120 Vol.71,No.1
1092-2172/07/$08.00(cid:1)0 doi:10.1128/MMBR.00033-06
Copyright©2007,AmericanSocietyforMicrobiology.AllRightsReserved.
Biosynthesis of Ether-Type Polar Lipids in Archaea and
Evolutionary Considerations
Yosuke Koga* and Hiroyuki Morii
DepartmentofChemistry,UniversityofOccupationalandEnvironmentalHealth,Kitakyushu807-8555,Japan
INTRODUCTION.........................................................................................................................................................97
ISOPRENOIDBIOSYNTHESIS................................................................................................................................99
MVAPathwayfromAcetyl-CoAtoDMAPP..........................................................................................................99
PolyprenylDiphosphateSynthesis.......................................................................................................................101
FORMATIONOFTHEG-1-PBACKBONE..........................................................................................................102 D
o
InVivoEvidence.....................................................................................................................................................102 w
DirectParticipationofG-1-P................................................................................................................................103 n
DiscoveryofG-1-PDehydrogenase(EC1.1.1.261)............................................................................................103 lo
InterpretationoftheInVivoPhenomenaObservedinHalobacterium...........................................................104 a
d
PhylogeneticRelationshipsandMolecularMechanismsofG-1-PDehydrogenases.....................................105 e
G-1-PFormationinSulfolobus..............................................................................................................................105 d

ETHERBONDFORMATION..................................................................................................................................105 fr
o
FirstEtherBondFormation(G-1-P:GGPPGeranylgeranyltransferase;GGGPSynthase;EC2.5.1.41).105 m
SecondEtherBondFormation(GGGP:GGPPGeranylgeranyltransferase;DGGGPSynthase;EC
h
2.5.1.42)................................................................................................................................................................108 t
t
ATTACHMENTOFPOLARHEADGROUPS......................................................................................................108 p
:
InVivoPulse-LabelingExperiments....................................................................................................................108 //
m
CDP-ArchaeolSynthase(CTP:2,3-di-O-Geranylgranyl-sn-Glycero-1-PhosphateCytidyltransferase).........108
m
ArchaetidylserineSynthase(CDP-2,3-Di-O-Geranylgeranyl-sn-Glycerol:L-Serine b
O-Archaetidyltransferase)..................................................................................................................................109 r
.
HomologySearchfortheCDP-AlcoholPhosphatidyltransferaseFamily.......................................................110 a
s
ArchaetidylethanolamineSynthesis......................................................................................................................110 m
1L-myo-Inositol-1-Phosphate Synthase (EC 5.5.1.4) and 1L-myo-Inositol-1-PhosphatePhosphatase .
o
(EC 3.1.3.25)........................................................................................................................................................111 r
g
HydrogenationofUnsaturatedIntermediates....................................................................................................113 /

GlycolipidSynthesis...............................................................................................................................................113 o
n
BIOSYNTHESISOFTETRAETHERPOLARLIPIDS.........................................................................................113
J
SimpleKineticStudiesbetweenDietherandTetraether(Polar)Lipids........................................................114 a
InhibitionofTetraetherLipidSynthesis.............................................................................................................114 n
u
PossibleInvolvementofRadicalIntermediates?................................................................................................114 a
EVOLUTIONOFMEMBRANELIPIDANDDIFFERENTIATIONOFARCHAEAANDBACTERIA........116 ry
HybridNatureofthePhospholipidSynthesisPathway....................................................................................116 4
DifferentiationofArchaeaandBacteriaCausedbySegregationofEnantiomericMembranePhospholipid, ,
2
andtheEvolutionofPhospholipids....................................................................................................................116 0
FUTURESUBJECTSOFLIPIDBIOSYNTHESISINARCHAEAANDCONCLUDINGREMARKS.........117 1
9
ACKNOWLEDGMENTS...........................................................................................................................................118
b
REFERENCES............................................................................................................................................................118 y

g
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INTRODUCTION points of view. Throughout this period, a great number of
s
novel and unique structures of archaeal polar lipids were re- t
The concept of Archaea was proposed on the basis of phy-
ported and reviewed (19, 47, 49, 52, 53). Regarding these
logenetic analysis of small-subunit rRNA sequences (109). In
structures,werecognizedfourstructuralcharacteristicsofar-
addition to the rRNA sequences, a number of biochemical
chaeallipidsthataredistinctfromtheirbacterialandeucaryal
properties of Archaea that are distinct from those of Bacteria
counterparts(Fig.1).Theyaresummarizedasfollows.(i)The
and Eucarya supported the concept that these three domains
stereostructure of the glycerophosphate backbone: hydrocar-
arethemostbasictaxaofalllivingorganisms.Membranepolar
bon chains are bound at the sn-2 and sn-3 positions of the
lipids are some of the most remarkable features among the
glycerolmoietyinarchaeallipids,whilebacterialandeucaryal
distinct characteristics of archaea. Archaeal lipids have been
lipidshavesn-1and2-diradylchains.Thatis,theglycerophos-
studied since the 1960s from the structural and biosynthetic
phate backbone of archaeal phospholipids is sn-glycerol-1-
phosphate(G-1-P),whichisanenantiomerofthesn-glycerol-
3-phosphate (G-3-P) backbone of bacterial and eucaryal
*Correspondingauthor.Mailingaddress:1-1Iseigaoka,Yahatanishi-
phospholipids. (ii) Ether linkages: hydrocarbon chains are
ku,Kitakyushu807-8555,Japan.Phone:[phone].Fax:[phone]-
9921.E-mail:[email protected]. bound to the glycerol moiety exclusively by ether linkages in
97
98 KOGA AND MORII MICROBIOL.MOL.BIOL.REV.
D
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FIG. 1. Characteristics of archaeal polar lipids. Archaeal phospholipids are characterized by (i) G-1-P backbone, (ii) ether bonds, (iii)
h
isoprenoid hydrocarbon chains, and (iv) bipolar tetraether lipids. Most of the polar head groups of phospholipids are shared by archaea and
t
bacteria. tp
:
/
/
m
m
archaeal lipids, in contrast to the situation for bacterial polar poration experiments of radioactive glycerol into lipids in b
lipids, most of which have ester linkages between fatty acids Halobacterium cutirubrum (later renamed Halobacterium sali- r.
a
and a glycerol moiety. (iii) Isoprenoid hydrocarbon chains: narum[104]),whichallowedexplorationofthemechanismof s
m
hydrocarbonchainsofpolarlipidsarehighlymethyl-branched theformationoftheenantiomericglycerophosphatebackbone.
.
isoprenoids in archaea, while their bacterial counterparts are However,onlyinvivostudieswerereportedoccasionallyinthe o
r
mostlystraight-chainfattyacids.(iv)Bipolartetraetherlipids: 20yearsafter1970.In1990,thefirstinvitrostudyofarchaeal g
/
bipolar lipids with a tetraether core are present in significant lipid biosynthesis was published (115). The enzymatic activity o
n
numbersinarchaealspecies.Thesetetraetherpolarlipidsspan of ether bond formation was reported for Methanobacterium

J
themembranetoformamembranemonolayer.Amongthese thermoautotrophicum strain Marburg (recently reclassified a
characteristics, the stereostructure of the glycerophosphate Methanothermobacter marburgensis [107]). In vitro studies of n
u
backboneisthemostspecifictoorganismsofeachdomainin themajorpathwayofpolarlipidbiosynthesisinarchaeahave a
r
terms of structure. It is the most crucial feature of archaeal been published in the 15 years since the in vivo studies were y

lipids of phylogenetic and evolutionary significance (see be- carried out during the preceding two decades (1970 to 1990). 4
,

low). However, the enantiomeric difference appears to be in- To the best of our knowledge, no review specifically dealing 2
0
significantforthephysicochemicalpropertiesofthelipidmem- with the in vitro biosynthesis of archaeal lipids has yet been 1
brane of archaea, because the enantiomers are equivalent in published. Recently, phylogenetic analyses and evolutionary 9

b
physicochemicalpropertiesexceptforchirality.Theetherlink- considerationsoftheenzymesforlipidbiosynthesiswerecar-
y
ages, isoprenoid chains, and bipolar tetraether lipids are sig- ried out (10), but polar lipid biosynthesis was not discussed. g
nificant for the physicochemical properties of archaeal lipid Although previous reviews mainly dealing with archaeal lipid u
e
membranes,forexample,theirphasebehaviorandtheperme- structures 10 years or more ago partly described biosynthetic s
t
abilityofthemembranes(52). aspects,thosewererestrictedtoinvivostudies(48,53).There-
In contrast to the four differences, most of the polar head fore, this is the first review that focuses mainly on in vitro
groupsofphospholipidsaresharedbyorganismsofthethree biosynthetic studies of archaeal polar lipids. The present re-
domains,withminorexceptions.Ethanolamine,L-serine,glyc- viewassemblescollectedknowledgeontherecentprogressin
erol,myo-inositol,andevencholinearefoundthroughoutthe in vitro studies of the biosynthesis of the four characteristic
three domains as phosphodiester-linked polar head groups in structuresofpolarlipidsinarchaeaalongwiththeprecedingin
phospholipid (31, 52, 53). Some minor unique polar groups, vivo studies (isoprenoid biosynthesis, sn-glycerol-1-phosphate
such as di- and trimethylaminopentanetetrols (30), glucos- formation, ether bond formation, phospholipid polar head
aminyl-myo-inositol (78), and glucosyl-myo-inositol (71), are groupattachment,glycolipidsynthesis,andtetraetherlipidfor-
foundinalimitednumberofspeciesofarchaea. mation),anditalsoprovidesacomparisonwithbacterialpolar
The biosynthetic mechanisms by which these characteristic lipidbiosynthesis.
lipidstructuresareformedhavebeenasubjectofinterestfor Inspiteofthelimitednumberofenzymaticstudiesofpolar
a long time. As early as 1970, the first metabolic study on lipidbiosynthesisinarchaea,quiteanumberofgenesrelevant
archaeal lipids was reported (50). It comprised in vivo incor- tolipidbiosynthesishavebeenfoundinthegenomesequences
VOL.71,2007 BIOSYNTHESIS OF ARCHAEAL POLAR LIPIDS 99
TABLE 1. Nomenclaturalchangesinarchaeadiscussedinthepresenttext
Originalname Strain Namemostrecentlyproposed Reference
Caldariellaacidophila Sulfolobussolfataricus 116
Halobacteriumcutirubrum Halobacteriumsalinarum 104
Halobacteriumhalobium Halobacteriumsalinarum 104
Halobacteriummediterranei Haloferaxmediterranei 102
Halobacteriumvallismortis Haloarculavallismortis 102
Methanobacteriumthermoautotrophicum (cid:2)H Methanothermobacterthermautotrophicus 107
Methanobacteriumthermoautotrophicum Marburg Methanothermobactermarburgensis 107
Methanobacteriumthermoformicicum SF-4 Methanothermobacterwolfeii 107
Methanococcusigneus Methanotorrisigneus 108
Natronobacteriumpharaonis Natronomonaspharaonis 45
Pseudomonassalinaria Halobacteriumsalinarum 32
D
o
ofarchaea.Homologysearchesforenzymesthatareexpected mental question. This was first evidenced by in vivo incorpo- w
n
to be involved in archaeal polar lipid biosynthesis have re- ration experiments of exogenously supplied, isotopically la-
lo
vealedmanyhomologs,whichhavebeenusedforthedetection beledacetateintoisoprenoidchains.When[13CH ]acetatewas a
3
of activities of unknown enzymes and for the evolutionary incorporatedintoisoprenoidmoietiesofpolarlipidsinMeth- d
e
analysisofthepolarlipidsyntheticpathway.Theevolutionary anospirillumhungatei(28)andSulfolobussolfataricus(18),13C d

aspects of polar lipid biosynthesis are also discussed in this appearedatthepositionsofmethylcarbons(C-17,C-18,C-19, fr
o
review. Because polar lipids are the principal and essential and C-20) and carbons (C-2, C-4, C-6, C-8, C-10, C-12, C-14, m
constituentsofthecytoplasmicmembranesofallcells,funda- and C-16). In contrast, 13C was detected at the methine car-
h
mental differences in polar lipid structure likely reflect the bons (C-3, C-7, C-11, and C-15) and at the carbon positions tt
p
history of diversification of fundamental cellular lines. The C-1,C-5,C-9,andC-13when[13COOH]acetatewasfedtothe :
/
uniformityanddiversityofthemembranepolarlipidstructures same archaeal cultures. These results are consistent with the /m
of archaea and bacteria are discussed. The nomenclature for positionsexpectedfromlabelingviatheMVAisoprenoidsyn- m
archaealpolarlipidsproposedbyNishiharaetal.(77)isused thesis pathway from three molecules of acetyl-CoA known b
r
throughout this paper. Scientific names of archaea as they in eucarya (Fig. 3). However, Ekiel et al. (29) reported that .a
appear in the original references or have been changed most in Halobacterium cutirubrum, neither [13COOH]acetate nor sm
recentlyareusedinthistext.Thechangesinnomenclatureof [13CH ]acetate was incorporated into branch-methyl and .
3 o
thearchaeaappearinginthepresenttextarelistedinTable1. methine carbons in phytanyl chains; instead, they were derived r
g
fromlysine.Because[14C]mevalonicacidwasefficientlyincor- /
o
ISOPRENOIDBIOSYNTHESIS poratedintolipids,andcarbonsatpositionsotherthanbranch n

and methine were labeled by acetate, a new route for the J
The hydrocarbon portions of archaeal diether lipids are a
formationofMVAfromthetwomoleculesofacetate(acetyl- n
exclusively isoprenoids (C20 phytanyl, C25 sesterterpenyl or CoA) and lysine was presumed. However, this result has not u
farnesylgeranyl groups). While isoprenoids are found only in a
archaea as a component of polar lipids, more than 25,000 been adequately evaluated because no specific biochemical ry
reactionwasdelineated. 4
naturally occurring isoprenoid derivatives are known to exist ,
ItwasreportedthatfarnesolinhibitsthegrowthofHaloferax
throughouttheorganismsofthethreedomains.Asisoprenoid 2
volcaniiandlipidsynthesis(98).Theinhibitionisattributedto 0
biosynthesisisamoreordinarysubjectthantheotheraspects 1
an inhibition of acetate incorporation into lipids, which is re- 9
of the biosynthesis of archaeal polar lipids, the subject has
covered by the addition of MVA. Farnesol did not affect the b
beenmoreextensivelystudiedandreviewed(9,93).Therefore, y
incorporation of MVA. Accordingly, it was concluded that
this section is limited to Archaea-specific topics of isoprenoid g
farnesolinhibitsthesynthesisofMVAfromacetate.Thisphe- u
biosynthesis. e
nomenonsuggeststheexistenceofaregulatorymechanismof s
isoprenoid synthesis by farnesol in this organism. It is not t
MVAPathwayfromAcetyl-CoAtoDMAPP
knownwhetherfarnesolinhibitionoccursinotherarchaea.
Theisoprenoidbiosyntheticpathwayisdividedintotwosec- AnearlyinvitroworkonC40carotenesynthesisinHalobac-
tions. The first half is the synthesis from acetyl-coenzyme A teriumcutirubrumalsoshowedthatisoprenoidsaresynthesized
(CoA) to isopentenyl diphosphate (IPP) and dimethylallyl fromMVAviaIPP,eventhoughcarotenesarenotcomponents
diphosphate (DMAPP), and the second half is the synthesis of polar lipids (57). In vitro assays of enzyme activities of the
of polyprenyl diphosphate from the two C units (IPP and pathwaysupportedtheconclusionledbytheinvivoevidence.
5
DMAPP). For IPP synthesis, two independent pathways are 3-Hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase, a
known:theclassicalmevalonate(MVA)pathway(Fig.2)and keyenzymeoftheMVApathway,wasdetected,purified,and
amorerecentlydiscoveredMVA-independent(1-deoxy-D-xy- characterizedfromHaloferaxvolcanii(6)andSulfolobussolfa-
lulose5-phosphate[DOXP])pathway(92).Theformerisusu- taricus(7).TheHaloferaxenzymewasfoundtobesensitiveto
ally found in eucarya and the latter in bacteria, algae, and lovastatin, an inhibitor of HMG-CoA reductase in mammals.
higher plants. Which pathway is functional in archaea for the ThegenesencodingHMG-CoAreductaseinHaloferaxvolcanii
formation of the isoprenoid chains of polar lipids is a funda- andSulfolobussolfataricuswereclonedandexpressedinEsch-
100 KOGA AND MORII MICROBIOL.MOL.BIOL.REV.
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4
,

2
0
1
9

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y

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FIG. 2. MVApathwayforsynthesisofisopentenyldiphosphateanddimethylallyldiphosphate.1,acetyl-CoAacetyltransferase;2,HMG-CoA u
e
synthase;3,HMG-CoAreductase;4,MVAkinase;5,phosphomevalonatekinase;6,diphosphomevalonatedecarboxylase;7,isopentenyldiphos-
s
phateisomerase;8,hypotheticalphosphomevalonatedecarboxylase;9,isopentenylphosphatekinase.TheclassicalMVApathwayproceedsfrom t
reaction1throughreaction7viareactions5and6,whileamodifiedMVApathwaygoesthroughreactions8and9(33).PandPPinthestructural
formulaarephosphateandpyrophosphate,respectively.
erichia coli cells. Sulfolobus HMG-CoA reductase showed fulgidus, which is a class II enzyme and is hypothesized to be
morethan40%similaritytoeucaryalhomologs(7).Thepuri- laterallytransferredfrombacteria(9).
fied enzymes from both archaeal species displayed similar ki- The seven enzymes and their genes in the MVA pathway
netic properties to the mammalian enzyme. The HMG-CoA from acetyl-CoA to DMAPP are most completely character-
reductasesfromvariouseucaryaandbacteriaweredividedinto ized for yeast. When a BLAST search was performed with
twoclassesbasedonaminoacidsequences:classIincludesthe thesesequencesasqueries,threeenzymeshadnomatchinany
eucaryal enzymes, and class II includes mainly the enzymes archaealgenome(93).Orthologousgenesforthefourenzymes
from bacteria (8). Archaeal HMG-CoA reductases belong to in the first half of the MVA pathway (acetoacetyl-CoA syn-
class I, with the exception of the enzyme from Archaeoglobus thase, HMG-CoA synthase, HMG-CoA reductase, and MVA
VOL.71,2007 BIOSYNTHESIS OF ARCHAEAL POLAR LIPIDS 101
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FIG. 3. Expectation of 13C incorporation from [13CH3]acetate into GGPP via the MVA pathway (18, 28). PP in the structural formula is d
pyrophosphate.
f
r
o
m

h
kinase) were detected, while the three orthologs for phos- found in the whole-genome sequences of many archaea.
t
t
phomevalonate kinase, diphosphomevalonate decarboxylase, Thus,ithasbeenverifiedthatinarchaeatheMVApathway p
:
andIPPisomerasewerenot.SmitandMushegian(93),based is responsible for the formation of the two essential C //
5 m
on an analysis of the sequence motifs of the known enzymes, intermediates (IPP and DMAPP) for the biosynthesis of
m
foundcandidateenzymesforthemissingstepsinsuperfamilies isoprenoids, and no evidence for the DOXP pathway has b
ofgalactokinase,nucleosidemonophosphatekinase,andMutT been obtained. r.
a
protein(8-oxo-7,8-dihydro-dGTPpyrophosphohydrolase[62]) s
m
in archaeal genomes. They inferred three genes encoding the
PolyprenylDiphosphateSynthesis .
three enzymes based on linkages, phylogenetic relationships, o
r
andsequencesimilarities.Ontheotherhand,Growchouskiet The involvement of polyprenyl diphosphate in polar lipid g
/
al.(33)haveidentifiedisopentenylphosphate(IP)kinaseasan synthesiswassupportedbytheinhibitionofgrowthandpolar o
MJ0044geneproductthatisoneoftheisoprenebiosynthesis lipidsynthesisinHalobacteriumcutirubrumbybacitracin.The n

J
genesofthehyperthermophilicmethanoarchaeonMethanocaldo- inhibitoryeffectisattributedtoitscomplexingwithpolyprenyl a
coccus jannaschii. The presence of IP kinase, in conjunction diphosphates(67). n
u
with the absence of phosphomevalonate kinase and diphos- DMAPP is consecutively condensed with several IPP mole- a
r
phomevalonatedecarboxylase,ledthemtoinferthatanalter- cules by prenyltransferase (polyprenyl synthase). The products y

nativeroutefortheformationofIPPmaybeoperatinginthis aregeranyl(C ),farnesyl(C ),geranylgeranyl(C ),andfarne- 4
10 15 20 ,
organism and some related archaea. Their alternative route sylgeranyl(C )diphosphate(Fig.4).Theproductsareallinthe 2
25 0
includesformationofIPfromphosphomevalonatebyphospho- 1
mevalonate decarboxylase and conversion of IP to IPP by IP 9

b
kinase.Theformerenzymeisspeculativeandneedsbiochem-
y
icalconfirmation.Thus,modificationoftheMVApathwayisa g
possiblecandidatefortheIPPsyntheticmechanism,atleastin u
e
sevenspeciesofarchaea. s
t
AlthoughIPPisomeraseisnotessentialforE.coli,inwhich
IPPissynthesizedviatheDOXPpathway(34),IPPisomerase
is essential for organisms with the MVA pathway to form
DMAPP, which is the starting allylic C precursor for poly-
5
prenyldiphosphatesynthesis.IPPisomeraseactivity,however,
could not be detected in any archaea until 2004, when genes
fromtwoarchaealspecies,Methanothermobacterthermauto-
trophicus (2) and Sulfolobus shibatae (110), homologous to the
Streptococcussp.strainCL190type2IPPisomerasegenefni
(46), were cloned and expressed in E. coli cells. This new
type of IPP isomerase requires NAD(P)H and Mg2(cid:1) and is
strictly dependent on flavin mononucleotide without a net
redoxchange(37).Thepreviouslyknowntype1IPPisomer- FIG. 4. Synthetic pathway of polyprenyl diphosphate. PP in the
ase does not require these coenzymes. fni homologs are structuralformulaispyrophosphate.
102 KOGA AND MORII MICROBIOL.MOL.BIOL.REV.
trans form. On the other hand, cis-polyprenyl synthase has also thatdistinguishesarchaeafrombacteriaandeucarya.G-1-Pis
beencharacterized(39);cis-polyprenyldiphosphateisnotapre- the enantiomer of bacterial G-3-P. Formation of the G-1-P
cursor of membrane polar ether lipids but is possibly a glycosyl backboneofpolarlipidswasinvestigatedinvivoin1970.Kates
carrierprecursor.Therefore,wedonotdiscusscis-prenyldiphos- et al. (50) first reported radiolabeled glycerol incorpora-
phate synthase in the present paper. The mechanism of each tion into an extremely halophilic archaeon, Halobacterium
condensing reaction is quite similar; i.e., the allyl diphosphate cutirubrum. When [1(3)-14C]glycerol and [2-3H]glycerol were
(DMAPP)servesasanacceptorofIPP.Theproductofthefirst incorporatedintoaglycerolmoietyofpolarlipids,the3H/14C
reactionisalsoanallyldiphosphate,whichcaninturnreactwith ratiowasgreatlyreducedcomparedwiththeratioofthepre-
anothermoleculeofIPPtoformaproductthatisasingleC unit cursors, while [1(3)-3H]glycerol was incorporated into lipids
5
longer.Theseenzymeswithdefiniteproductspecificitycansyn- without a loss of 3H. Kates et al. also detected glycerophos-
thesizeproductsofshorterchainlengths.Polyprenylsynthasesare phate dehydrogenase and glycerol kinase activities in this
commonthroughoutthethreedomainsoflivingorganisms,i.e., organism, and these were both G-3-P specific (106). They
yeast,Archaea,andBacteria.InArchaea,geranylgeranyldiphos- postulated that retention of 3H at the 1 position of glycerol
phate(GGPP)synthasewasfoundinMethanobacteriumthermo- excluded the involvement of dihydroxyacetonephosphate D
o
formicicum SF-4 (100) (reclassified Methanothermobacter wolfeii (DHAP), which undergoes keto-aldehyde isomerization be- w
[107]),Methanothermobactermarburgensis(12),Sulfolobusacido- tweenDHAPandD-glyceraldehyde-3-phosphate(GAP)bythe n
caldarius(85),andPyrococcushorikoshiiOT-3(64).GGPPsyn- high level of activity of triose phosphate isomerase. These lo
a
thasealsoproducesfarnesyldiphosphatefromDMAPPandIPP. observations led to the hypothesis that exogenously supplied d
e
ThecatalyticmechanismofaGGPPsynthasefromMethanother- glycerol was oxidized at the 2 position, forming dihydroxyac-
d
mobacterthermoautotrophicumSF-4wasshowntobeanordered- etone(DHA),onwhichoneetherlinkageisthenformedatthe f
r
sequential Bi-Bi mechanism. Potassium ions stimulate the en- 1position.ThealkylationofDHAisfollowedbyrereductionat o
m
zyme activity by expediting binding of the substrates (99). the2position,asecondetherbondformation,andphosphor-

h
Farnesylgeranyl diphosphate synthase was found in Na- ylation. At the rereduction stage, the stereoconfiguration of
t
t
tronobacteriumpharaonis(97)(laterreclassifiedNatronomo- thenewhydroxylgroupisformedsothatthefirstetherbond p
:
nas pharaonis [45]) and Aeropyrum pernix (101), whose polar shouldbelocatedatthesn-3position.G-1-P-formingenzymes //
m
lipidscontainC farnesylgeranylchains(20,71).Thegeneen- (glycerophosphate dehydrogenase and glycerol kinase) would
25 m
coding GGPP synthase (idsA) was cloned, identified, and ex- not be involved in this pathway. Instead, glycerol dehydroge- b
pressedfromMethanothermobactermarburgensis(11),Sulfolo- nase activity was detected in the cell extract of the same or- r.
a
bus acidocaldarius (85, 87), and Pyrococcus horikoshii (64). ganism(4)(theorganismcalledPseudomonassalinariaatthat s
m
These enzymes are characterized by aspartate-rich motifs in time [1954] was later renamed Halobacterium cutirubrum and
.
their sequences, which are commonly found in polyprenyl thenHalobacteriumsalinarum[32]).However,theoccurrence o
r
transferases. ofglyceroldehydrogenasewasspeciesdependentinthegenus g
/
As the molecular mechanism for regulation of the chain Halobacterium.WhileHalobacteriumsalinarum,Halobacterium o
n
lengthofthepolyprenylproductsofGGPPsynthaseofbacte- cutirubrum,andHalobacteriumhalobium(thesewerelaterre-

J
riaandarchaeahasalreadybeenreviewedbyLiangetal.(61), classified as the same species [104]) have high activities of a
and that article discussed the mechanism of chain length de- glyceroldehydrogenase,Halobacteriummediterranei(Haloferax n
u
terminationinSulfolobusacidocaldariusbyOhnumaetal.(82– mediterranei[102])andHalobacteriumvallismortis(Haloarcula a
r
84),alongwithbacteriaandeucarya,thissubjectisnolonger vallismortis [102]) do not (91). If the speculated pathway is y

dealtwithinthisreview. actually operating in halobacterial cells, all of these species 4
,

A salvage pathway for polyprenols was detected in Sulfo- musthavethisenzyme. 2
0
lobusacidocaldarius,inwhichgeranylgeraniolandgeranylgera- Kakinuma et al. (44) conducted similar experiments using 1
nyl monophosphate were phosphorylated with ATP by sepa- [2H]glycerol and nuclear magnetic resonance (NMR) detec- 9

rateenzymes(86). tion.Theysynthesizedposition-specificlabeledglycerol.2Hof b
y
Insummary,archaealisoprenoidsynthesisproceedsviathe [sn-1-2H]glycerol supplied to a culture of Halobacterium ap- g
classical MVA pathway that is shared with Eucarya , or its pearedatthesn-3positionoflipidglycerol,whilethe2Hofthe u
e
modifiedform;however,thearchaealMVApathwayisamo- [sn-3-2H]glycerolprecursorwasincorporatedintothesn-1po- s
t
saiccomposedoftheenzymescommontoArchaeaandEucarya, sitionofthelipid.Thisimpliesthataprochiralglycerolmole-
alongwithenzymesuniquetoArchaeaandBacteria.Inpartic- culeisinvertedduringlipidbiosynthesis.Basedonthisresult,
ular,type2IPPisomeraseandIPkinase,whicharethelatter they proposed that an asymmetrical intermediate should be
enzymes,areremarkablynovel.Thisillustratesthatelucidation involved in lipid biosynthesis, and the involvement of DHAP
not only of a metabolic pathway but also of the specifically instead of DHA was suggested to be the candidate for the
relevant enzymes is important for obtaining a deeper insight asymmetrical intermediate. Kakinuma’s pathway of glycerol
intothebiochemistryoflipidmetabolism. incorporation into lipid is as follows: glycerol 3 G-3-P 3
DHAP 3 alkyl DHAP 3 alkyl G-1-P 3 dialkyl G-1-P (ar-
chaetidicacid).ThismodelissimilartotheKatesmodelbutis
FORMATIONOFTHEG-1-PBACKBONE differentatthephosphorylationstepinthepathway.Enzymes
thatalkylateDHAParepresentinmammaliancells(103)but
InVivoEvidence
havenotbeenfoundinanyarchaea(seebelow).Theyincluded
Thedifferenceinthestereoconfigurationoftheglycerophos- G-3-PbutnotG-1-Pintheirproposal,sinceKatesetal.(106)
phatebackboneisoneofthemostremarkablecharacteristics reported that the glycerophosphate-forming enzyme of the
VOL.71,2007 BIOSYNTHESIS OF ARCHAEAL POLAR LIPIDS 103
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FIG. 5. Three possible reactions for the direct formation of G-1-P: A, reduction of D-GAP; B, reduction of DHAP; C, phosphorylation of a
r
glycerol(G-1-Pforming).Enzyme1,glycerolkinase(G-3-Pforming);enzyme2,G-3-Pdehydrogenase;enzyme3,triosephosphateisomerase; y

enzyme4,G-1-Pdehydrogenase.Reaction5,etherlipidsynthesis.FDP,fructosediphosphate. 4
,

2
0
1
organism was specific to G-3-P. Although the pathway was canformG-1-P.ThesecondpossibilityfortheG-1-P-forming 9

thought to be unnatural because of the presence of unneces- reaction was reduction of DHAP at the 2 position (Fig. 5B). b
y
saryorfruitlessoxidationandrereductionoftheglycerolmoi- ThisreactionissimilartotheG-3-Pdehydrogenasereaction g
etyatthesn-2positioninthepathway,noevidenceagainstthe except for the stereospecificity. The last possibility was di- u
e
pathway was presented until Poulter et al. (115) reported a rectphosphorylationofglycerolatthesn-1positionbyATP s
t
G-1-P-specificetherbond-formingenzyme. (Fig. 5C).
DirectParticipationofG-1-P DiscoveryofG-1-PDehydrogenase(EC1.1.1.261)
Poulter et al. (115) investigated in vitro ether bond forma- Nishiharaetal.(75)detectedglycerophosphate-formingac-
tioninMethanothermobactermarburgensisandfoundaG-1-P- tivity from DHAP in a cell-free homogenate of Methanother-
specific ether bond-forming enzyme (see the next section for mobacterthermautotrophicus.Thehomogenatealsocontained
details).ThissuggestedthatG-1-Pwasdirectlyinvolvedinthe extremelyhightriosephosphateisomeraseactivity,whichcat-
formationofetherlipids.Therefore,directformationofG-1-P alyzes interconversion between GAP and DHAP. Therefore,
cametobethefocusasthenextproblem.Threereactionswere glycerophosphatewasapparentlyformedalsofromGAPwhen
considered to be candidate mechanisms for G-1-P formation GAPandNADHwereincubatedwiththeunfractionatedho-
(Fig.5).OnewasthereductionofGAPatthe1position(Fig. mogenate.Atthisstage,thefirstandsecondpossibilitiescould
5A). Because D-GAP has the same configuration as that of notbediscriminated.GlycerophosphateformationfromGAP
G-1-Patthe2position,simplereductionofitsaldehydegroup was proven to be comprised of combined reactions of triose
104 KOGA AND MORII MICROBIOL.MOL.BIOL.REV.
D
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FIG. 6. Glycerolmetabolismandlipidbiosynthesisinarchaea.Thereactionsindicatedwithopenarrowsarethecatabolicpathwayofglycerol
h
inheterotrophicarchaea.Reactionsshownwithclosedarrowsarethesyntheticpathwayofphospholipidinarchaea(79).
t
t
p
:
/
/
m
phosphateisomeraseandglycerophosphatedehydrogenaseby etal.(79)discussedthefactthatonlyheterotrophicarchaea
m
fractionation of the two enzymes with a DEAE-cellulose col- contain a G-3-P-specific enzyme set (both G-3-P dehydroge- b
umn.Becausethefractionsofcell-freehomogenatesincluding nase and G-3-P-forming glycerol kinase), as follows (Fig. 6). r.
a
notriosephosphateisomeraseactivitydidnotexhibitglycero- Whentheseheterotrophicarchaeautilizeglycerolasacarbon s
m
phosphate formation activity from GAP, the possibility of an or energy source, they convert glycerol to G-3-P but not to
.
enzyme catalyzing direct formation of glycerophosphate from G-1-P. The produced G-3-P is further metabolized to DHAP o
r
GAP was excluded. The activity that catalyzes glycerophos- byG-3-Pdehydrogenase,andDHAPentersthecentralmeta- g
/
phate formation from DHAP was purified to homogeneity bolicpathwayafterconversiontoGAP.Inthisscenario,G-3-P o
n
(74). The reaction product from DHAP was confirmed to be isusedforglycerolcatabolism.G-1-Pisusedforlipidbiosyn-
J
G-1-P,andG-1-PbutnotG-3-Pwasoxidizedinthepresence thesis in archaea. DHAP is the crossing point of both path- a
ofNAD[DHAP(cid:1)NAD(P)H3G-1-P(cid:1)NAD(P)].NADH n
ways. These two pathways hence might confuse the results of u
acted as a coenzyme, while NADPH was also shown to be a
the in vivo incorporation experiments performed by Kates et r
activebutatasignificantlylowerlevelthanNADH.Therefore, y
al. and Kakinuma et al. Apparent inversion of the prochiral
this enzyme was established as a new enzyme of G-1-P dehy- 4
stereostructure of glycerol should be seen only in the case of ,

drogenaseandhasbeendesignatedEC1.1.1.261. 2
the incorporation of exogenously supplied glycerol into lipids 0
inheterotrophicarchaeathatisnotessentialforlipidsynthesis 1
9
InterpretationoftheInVivoPhenomenaObserved itself. Although Kates et al. regarded retention of H at the 1 b
inHalobacterium position of glycerol as evidence against the involvement of y

g
G-1-P-forming activity has been detected in cell extracts of DHAP because DHAP is in equilibrium with GAP, the ab- u
sence of any loss of the 3H of [1-3H]glycerol during incorpo- e
all of the archaeal species so far examined, such as Methano- s
rationintolipidsmightbeinterpretedbythelargeequilibrium t
thermobacterthermautotrophicus,Methanosarcinabarkeri,
constantbetweenDHAPandGAP(K(cid:3)[DHAP]/[GAP](cid:3)22
Halobacterium salinarum, Pyrococcus furiosus, Pyrococcus sp.
[5]). Because the equilibrium greatly favors DHAP, it is con-
strain KS8-1, and Thermoplasma acidophilum strain HO-62
ceivable that the DHAP produced from glycerol via G-3-P is
(79, 111). G-3-P was also formed from DHAP by incubation
with NADH or NADPH and cell extracts from some of the quickly reduced to G-1-P without substantial conversion to
species (Methanothermobacter thermautotrophicus, Halobacte- GAP. Alternatively, there might be separate pools of DHAP
riumsalinarum,Pyrococcussp.strainKS8-1,andThermoplasma forlipidsynthesisandcatabolism.
acidophilumstrainHO-62).G-1-Pdehydrogenaseactivitywas Kakinumaetal.(42)alsoreportedthatwhenD-[6,6-2H]glu-
alsoconfirmedinrecombinantproteinsfromAeropyrumpernix cosewasfedtoaHalobacteriumculture,deuteriumappeared
(36)andSulfolobustokodaii(54).G-1-Phasneverformedfrom at the sn-1 position of lipid glycerol. Because glucose is me-
glycerol and ATP, while G-3-P is formed from glycerol and tabolized via a modified Entner-Doudoroff pathway, carbons
ATP by cell extracts from Halobacterium, Pyrococcus, and at the 4, 5, and 6 positions of glucose are converted to the
Thermoplasma. These are all heterotrophs. Accordingly, the carbons1,2,and3oftheD-GAPthatisisomerizedtoDHAP.
thirdpossibilityforG-1-Pformationhasbeenexcluded.Nishihara Therefore, this result of Kakinuma is consistent with the in-
VOL.71,2007 BIOSYNTHESIS OF ARCHAEAL POLAR LIPIDS 105
volvement of G-1-P dehydrogenase proposed by Nishihara 3H]glycerol were incorporated into lipid glycerol without any
et al. loss of 3H in in vivo labeling experiments in Caldariella acid-
ophila (Sulfolobus solfataricus [116]) (21). Kakinuma et al.
confirmed this result by using stereospecifically labeled glyc-
PhylogeneticRelationshipsandMolecularMechanismsof
erolwith2Handreportednoinversionoftheglycerolmoiety
G-1-PDehydrogenases
of lipids during biosynthesis in Sulfolobus acidocaldarius (41).
The gene encoding G-1-P dehydrogenase of Methanother- Onepossibleexplanationfortheseresultsmaybedirectphos-
mobacter thermautotrophicus was cloned, sequenced, named phorylationofglycerolbyanunknownG-1-P-formingglycerol
egsA (51), and heterologously expressed in E. coli (81). The kinase.Onlyonestudy,presentedorallyatameeting,hasthus
deduced amino acid sequence of G-1-P dehydrogenase from farreportedtheproductofglycerolkinaseinS.acidocaldarius
Methanothermobacter thermautotrophicus is composed of 347 tobeG-3-P(81a).AlthoughthephenomenadescribedbyDe
amino acid residues with a molecular weight of 36,963. Ar- Rosa et al. and Kakinuma et al. remain to be explained, the
chaeal G-1-P dehydrogenase and bacterial G-3-P dehydroge- universality of the presence of G-1-P dehydrogenase was ver-
nasesharelittlesequencesimilarity,showingthattheybelong ified by the detection of G-1-P dehydrogenase in a species of D
o
todifferentenzymefamilies.Adatabasesearchdetectedopen Sulfolobus(S.tokodaii)(54). w
readingframes(ORFs)homologoustotheegsAgeneinall21 n
speciesofarchaeawhosewholegenomeshadbeensequenced lo
ETHERBONDFORMATION a
upuntilthattime,anditwasshownthatthearchaealenzyme d
e
exhibitedsequencesimilaritytoglyceroldehydrogenase,dehy- The pathways for biosynthesis of the ether-type and ester-
d
droquinate synthase, and alcohol dehydrogenase IV (16). A typephospholipidsfromarchaeaandbacteria,respectively,are f
r
phylogenetictreeofG-1-Pdehydrogenasesandtheirhomologs depictedinFig.8. o
m
was constructed (Fig. 7). Glycerol dehydrogenase, with coor-

h
dinatesavailableatthattime,wasshowntobecloselyrelated
FirstEtherBondFormation(G-1-P:GGPP tt
toG-1-Pdehydrogenase.Usingthestructureofglyceroldehy- p
Geranylgeranyltransferase;GGGP :
drogenase as the template, Daiyasu et al. (16) built a model Synthase;EC2.5.1.41) //m
structure of G-1-P dehydrogenase that predicts the following
m
structure and function characteristics of the enzyme: (i) the Etherlipidbiosynthesiswasstudiedbyinvivoincorporation b
chirality of the product, (ii) the requirement of the Zn2(cid:1) ion of several lipid components into Methanospirillum hungatei r.
a
fortheenzymereaction,(iii)thetransferofpro-Rhydrogenof cells(90).Archaeol(2,3-di-O-phytanyl-sn-glycerol)andcaldar- s
NADHduringtheenzymereaction,(iv)theputativeactivesite chaeol(2,3,2(cid:4),3(cid:4)-di-O-bisphytandiyl-di-sn-glycerol)wereincor- m
.
and the reaction mechanism, and (v) that G-1-P dehydroge- porated into mainly the nonpolar lipid fraction, and no o
r
nase does not share an evolutionary origin with G-3-P dehy- interconversion between them was found. Among the C g
20 /
drogenase from bacteria. The pro-R hydrogen transfer and polyprenols, fully unsaturated geranylgeraniol was most effi- o
Zn2(cid:1) requirement were experimentally verified (35, 55). It is ciently incorporated into polar lipids in intact cells of the n

J
knownthatG-3-Pdehydrogenasetransfersthepro-Shydrogen methanogenic archaeon. Monounsaturated phytol was poorly a
of NADH (23). Crystallographic data showed that pro-R ste- incorporated, and fully saturated phytanol was not incorpo- n
u
reospecificenzymesandpro-Sstereospecificenzymesbindthe rated. The results suggested that an ether bond was formed a
r
nicotinamideringofthecoenzymeattheoppositeorientation, from unsaturated prenyl precursors (90). This was confirmed y

andthisisapparentlythebasisfortheenzymestereospecificity byaninvitroenzymaticexperimentwithsonicatesofMethan- 4
,

(112). Therefore, it is assumed that G-1-P dehydrogenase othermobacter marburgensis cells (115). Ether bond formation 2
0
(pro-Rtype)andG-3-Pdehydrogenase(pro-Stype)havesym- is catalyzed by two prenyl transferases: one is responsible for 1
metrical ternary structures, at least in the coenzyme-binding formation of the first ether bond between the sn-3 hydroxyl 9

b
sites. G-1-P dehydrogenase in Aeropyrum pernix has been ki- group of G-1-P and GGPP (GGGP synthase), and the other
y
neticallystudied(36).TheenzymeusesNADHorNADPHas catalyzestheformationofthesecondetherbondatthesn-2 g
acoenzymeinDHAPreductionwithpreferenceforNADPH position to form di-O-geranylgeranyl-G-1-P (DGGGP, or u
e
but does not use NADP in G-1-P oxidation. This fact, along unsaturated archaetidic acid) (DGGGP synthase) (114). s
t
with the far lower K value for DHAP than for G-1-P (74), The first enzyme was found in sonic extracts of Methano-
m
suggeststhatG-1-Pdehydrogenaseisreallyfunctioninginthe thermobactermarburgensiscellsandHalobacteriumhalobium
direction of G-1-P formation from DHAP. A kinetic analysis cells(113).Theenzymeshowed30to40timeshigheractiv-
revealed that the catalytic reaction by the enzyme follows an ity to G-1-P than to G-3-P and did not react with DHAP.
orderedBi-Bimechanism(36). This evidence directly contradicts Kakinuma’s hypothesis.
Thus,G-1-Pdehydrogenasehasbeenestablishedattheen- GGGP synthase exhibited the same order of specificity of
zyme and genetic levels as a key enzyme responsible for the geranylgeranyl-, phytyl-, and phytanyl diphosphate as the
formation of the enantiomeric glycerophosphate backbone incorporation efficiencies of these prenyl alcohols exhibited
structureofarchaealphospholipids. in the in vivo incorporation experiments (115). Farnesyl
diphosphate could not serve as a substrate for GGGP syn-
thase (114).
G-1-PFormationinSulfolobus
GGGPsynthaseisasolubleenzyme,whilethesecondether
AnexamplethatcannotbeexplainedbytheG-1-Ppathway bond-forming enzyme is associated with the membrane frac-
isthecaseofSulfolobus.[U-14C,1(3)-3H]glyceroland[U-14C,2- tion (114). GGGP synthase was first purified to homogeneity
106 KOGA AND MORII MICROBIOL.MOL.BIOL.REV.
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FIG. 7. PhylogenetictreeofG-1-Pdehydrogenaseanditshomologs.Thesequenceisindicatedbythesourcenameofthesequencedatabase
(sp,SwissProt;pir,PIR;gb,GenBank;pdb,PDB)andtheidentificationcode.G1PDH,sn-glycerol-1-phosphatedehydrogenase;GDH,glycerol
dehydrogenase;DHQS,dehydroquinatesynthase;ALDH,alcoholdehydrogenasetypeIV.(Reprintedfromreference16bypermissionofOxford
UniversityPress.)