The unique features of glycolytic pathways in Archaea

16 Pages·2003·0.32 MB·English

Biochem.J.(2003)375,231–246(PrintedinGreatBritain) 231
REVIEW ARTICLE
The unique features of glycolytic pathways in Archaea
Corne´ H.VERHEES*1,Serve´ W.M.KENGEN*,JudithE.TUININGA*,GerritJ.SCHUT†,MichaelW.W.ADAMS†,
WillemM.DEVOS*andJohnVANDEROOST*2
*LaboratoryofMicrobiology,DepartmentofAgrotechnologyandFoodSciences,WageningenUniversity,HesselinkvanSuchtelenweg4,6703CTWageningen,TheNetherlands,
and†DepartmentofBiochemistryandMolecularBiology,UniversityofGeorgia,Athens,GA03602,U.S.A.
Anearlydivergenceinevolutionhasresultedintwoprokaryotic and novel enzymes in the archaeal variants of the Embden–
domains,theBacteriaandtheArchaea.Whereasthecentralmeta- Meyerhof and the Entner–Doudoroff pathways. By means of
bolic routes of bacteria and eukaryotes are generally well-con- integrating results from biochemical and genetic studies with
served, variant pathways have developed in Archaea involving recently obtained comparative and functional genomics data,
severalnovelenzymeswithadistinctcontrol.Aspectacularex- the structure and function of the archaeal glycolytic routes, the
ample of convergent evolution concerns the glucose-degrading participatingenzymesandtheirregulationarere-evaluated.
pathwaysofsaccharolyticarchaea.Theidentification,character-
ization and comparison of the glycolytic enzymes of a variety Keywords:Archaea,Embden–Meyerhofpathway,Entner–Dou-
of phylogenetic lineages have revealed a mosaic of canonical deroffpathway,glycolysis,glycolyticenzyme,polysaccharide.
INTRODUCTION include(i)identificationoffermentationendproductsbyHPLC
and 13C-NMR [4], (ii) identification of intermediates of sugar
Polysaccharidesareamajorsourceofcarbontosupporthetero-
metabolism following the conversion of 14C-labelled glucose
trophic growth in the three domains of life: Bacteria, Archaea
[5,6], (iii) enzyme-activity measurements in cell extracts [7],
andEukaryota.Theirutilizationgenerallyinvolvesextracellular
(iv) characterization of purified enzymes [8], and (v) molecular
hydrolysis of polysaccharides, uptake of oligosaccharides by
analysisofgenesencodingglycolyticenzymes,andcomparative
specifictransportersandtheirintracellularhydrolysistogenerate
genome analysis [9,10]. In the present review, the sugar meta-
hexoses (e.g. glucose, galactose, mannose and fructose) and
bolism of archaea is re-evaluated by integrating physiological,
pentoses(e.g.xyloseandarabinose).Subsequently,thesemono-
biochemical and genetic data with recent insights provided by
saccharidesarebeingoxidizedviaawell-conservedsetofcentral
comparativeandfunctionalgenomics.
metabolicpathways.
Avarietyofpathwaysisinvolvedinthedegradationofglucose
to pyruvate (Schemes 1A and 1B). The Embden–Meyerhof
SACCHAROLYTICARCHAEA
(EM) pathway, or glycolysis, is the general route for glucose
degradation in all domains of life. Some micro-organisms use Owingtotheeffortsofseveralresearchgroupsoverthepastde-
an alternative pathway for glucose degradation, i.e. the Entner– cade, insight is emerging into the sugar metabolism of archaea
Doudoroff(ED)pathway.Inaddition,someorganismsarecapable in general and that of hyperthermophiles in particular. Several
of alternative routes and bypasses in sugar degradation, include modified sugar-degrading pathways that are operational at ex-
the oxidative pentose phosphate (PP) pathway (also referred to tremelyhightemperatureshavebeenidentifiedinsomeofthese
asthehexosemonophosphatepathway)[1].Establishedpathways hyperthermophiles(reviewedin[10–14]).
for biosynthetic purposes include gluconeogenesis and the non- The Archaea comprise both autotrophic (e.g. methanogens,
oxidativePPpathway(Scheme1). sulphurreducersandsulphatereducers)andheterotrophicrepre-
Extensiveresearchoverseveraldecadeshasresultedindetailed sentatives[12].Mosthyperthermophilicarchaeaareheterotrophs
informationonthecompositionofsugarmetabolicpathwaysand that use polypeptides as carbon and energy sources. However,
the regulation thereof in bacteria and eukaryotes [2,3]. Because a growing number of archaeal species has been shown to be
mostarchaeahavebeendiscoveredonly1–2decadesago,studies saccharolyticaswell,andefficientgrowthhasbeenobservedon
addressingarchaealglycolyticpathwayswerefirstinitiatedinthe variouspoly-,oligo-andmono-saccharides(Table1).Amongthe
early1990s.Themostprogresshasbeenmadewithhyperthermo- archaea,majordifferencesoccurintheirenergy-transducingcapa-
philic archaea that grow optimally above 80◦C on a variety of city. Several archaea have been reported to perform respiration
sugars. The approaches taken to unravel these metabolic routes eitheraerobicallywithoxygenasaterminalelectronacceptor(e.g.
Abbreviationsused:AOR,aldehydeoxidoreductase;ED,Entner–Doudoroff;EM,Embden–Meyerhof;FBA,fructose-1,6-bisphosphatealdolase;FBP,
fructose-1,6-bisphosphatase;FOR,formaldehydeferredoxinoxidoreductase;GAP,glyceraldehyde3-phosphate;GAPDH,glyceraldehyde-3-phosphate
dehydrogenase;GAPN,NAD+-dependentGAPDH;GAPOR,glyceraldehyde-3-phosphateferredoxinoxidoreductase;GLK,glucokinase;KDG,2-keto-3-
deoxygluconate;KDPG,2-keto-3-deoxy-6-phosphogluconate;LDH,lactatedehydrogenase;ORF,openreadingframe;PEP,phosphoenolpyruvate;PFK,
phosphofructokinase;PFKB,minorPFK;PGI,phosphoglucoseisomerase;PGK,phosphoglyceratekinase;PGM,phosphoglyceratemutase;PP,pentose
phosphate;PPS,PEPsynthase;PTS,phosphotransferasesystem;PYK,pyruvatekinase;SIS,sugarisomerase;T ,optimumgrowthtemperature;TIM,
opt
triosephosphateisomerase.
1 Presentaddress:DSMResearch,A.Fleminglaan1,2613AXDelft,TheNetherlands
2 Towhomcorrespondenceshouldbeaddressed([email protected]).
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Scheme1 Glycolysisandgluconeogenesis
(A)GlucosedegradationinArchaeaviavariantsoftheEMandEDpathways.(B)Bacterial/eukaryalroutesofglucosecatabolism:EM,EDandoxidativePPpathway.(C)Gluconeogenesisinarchaeaandbacteria/eukaryaresultsinthereductionofpyruvatetofructose
6-phosphate(Fructose6P),whichmaybefurtherconvertedintoglucose6-phosphate(Glucose6P)orxylulose5-phosphate(Xylulose5P)viathenon-oxidativePPpathway.Abbreviatedmetabolitesin(A)–(C)are:GA,glyceraldehyde;Fructose1,6P,fructose
2
1,6-bisphosphate;PGP,2,3-bisphosphoglycerate;2PG,2-phosphoglycerate;3PG,3-phosphoglycerate;fdox,oxidizedferredoxin;fdred,reducedferredoxin.Keytoenzymes:(1)GLK;(2)PGI;(3)PFK;(4)FBA;(5)TIM;(6)GAPDH;(7)PGK;(6/7)GAPORandGAPN;
(8)PGM;(9)enolase;(10)PYK;(11)GlcDH/Glc-lac;glucosedehydrogenase/gluconolactonase;(12)G-hydr,gluconatedehydratase;(13)KDGald,KDGaldolase;(14)AldDH,glyceraldehydedehydrogenase;(15)Glykin,glyceratekinase;(16)KDGkin,KDG
kinase;(17)KDPGald,KDPGaldolase;(18)G6PDH/6PG-lac;glucose-6-phosphatedehydrogenase/6-phosphogluconolactonase;(19)GlcA-kin,gluconatekinase;(20)PG-hydr,6-phosphogluconatekinase;(21)PGDHDC,6-phosphogluconatedehydrogenase
(decarboxylase);(22)RPepi,ribulose-5-phosphate3-epimerase;(23)Tr.ketolase,transketolase;(24)PPS;(25)FBP.
GlycolysisinArchaea 233
Table1 Carbohydrate-metabolizingarchaea
Organism T (◦C) Metabolism Habitat Carbohydratesubstrates Pathway Reference
opt
Sulfolobales
Sulfolobussolfataricus 80 Aerobic Terrestrial Starch,dextrin,xyloglucan,maltose, ED* [13,29]
sucrose,lactose,glucose,xylose
Sulfolobusshibatae 81 Aerobic Terrestrial Starch,glucose,galactose,arabinose [13]
Sulfolobusacidocaldaricus 75 Aerobic Terrestrial Sucrose,lactose,glucose,galactose [13]
Thermoproteales
Thermoproteustenax 88 Anaerobic Terrestrial Starch,glycogen,glucose EM*/ED* [13]
Desulfurococcales
Desulfurococcusamylolyticus 90 Anaerobic Terrestrial Starch,glycogen EM* [5,13]
Desulfurococcussaccharovorans 85 Anaerobic Terrestrial Glucose [13]
Desulfurococcusmucosus 88 Anaerobic Terrestrial Starch [13]
Pyrodictiumabyssi 97 Anaerobic Marine Starch,glycogen,raffinose,lactose [13]
Thermococcales
Pyrococcusfuriosus 100 Anaerobic Marine Starch,pullulanan,glycogen,maltose,cellobiose, EM* [13,21];C.Verhees,
glucose,lactose†,melibiose† unpublishedwork
Pyrococcuswoesei 100 Anaerobic Marine Starch,glycogen,maltose,cellobiose EM* [13]
Pyrococcusglycovorans 95 Anaerobic Marine Starch,maltose,cellobiose,glucose [118]
Thermococcusceler 88 Anaerobic Marine Sucrose [119]
Thermococcusstetteri 75 Anaerobic Marine Starch EM* [13]
Thermococcuszilligii 75 Anaerobic Terrestrial Maltose EM* [56,120]
Thermococcuslitoralis 88 Anaerobic Marine Starch,maltose EM* [13,121]
Thermococcusprofundus 80 Anaerobic Marine Starch,maltose [13]
Thermococcushydrothermalis 90 Anaerobic Marine Cellobiose,maltose [122]
Thermococcusaggregans 88 Anaerobic Marine Starch,maltose [123]
Thermococcusguaymasensis 88 Anaerobic Marine Starch,maltose [123]
Thermococcuspacificus 85 Anaerobic Marine Starch [124]
Thermococcusfumicolans 85 Anaerobic Marine Maltose [125]
Thermococcusprofundus 80 Anaerobic Marine Starch,maltose [126]
Archaeoglobales
Archaeoglobusfulgidusstrain7324 83 Anaerobic Marine Starch EM* [48]
Thermoplasmales
Thermoplasmaacidophilum 59 Aerobic Terrestrial Glucose ED* [79]
Halobacteriales‡
Halococcussaccharolyticus 37 Aerobic Highsalt§ Arabinose,lactose,fructose,glucose EM/ED* [84,127]
Haloferaxmediteranei 35 Aerobic Highsalt§ Starch,lactose,sucrose,fructose,glucose EM/ED* [87,128,129]
Haloarculavallismortis 37 Aerobic Highsalt§ Fructose,glucose EM/ED* [129]
Halobacteriumsaccharovorum 37 Aerobic Highsalt§ Glucose ED* [130]
*ModificationinEM/EDpathway.
†Pyrococcusfuriosusdegradestheglucosemoietyoflactoseandmelibiose.Galactoseismainlysecretedinthemedium.
‡HalophilesuseanEM-likepathwayforthedegradationoffructoseandamodifiedEDpathwayforthedegradationofglucose.
§>12%(2M)NaCl.
Sulfolobus,Pyrobaculum,Thermoplasmaandhalophilicarchaea), degraded by specific extracellular glycosyl hydrolases to oligo-
oranaerobicallywithalternativeterminalelectronacceptors,such saccharides [20,21,25–29], which are subsequently transported
asnitrate(e.g.Pyrobaculum),sulphur(e.g.Thermoproteus),sul- intothecellbyeitherABC(ATP-bindingcassette)-typeorsecond-
phate(e.g.Archaeoglobus)orcarbondioxide(e.g.methanogens). ary transporters [23,30–35]. Sugar transport via the phospho-
In some organisms, the generation of membrane potential has enolpyruvate(PEP)-dependentphosphotransferasesystem(PTS)
neverbeendemonstratedexperimentally,andhencetheymayde- isverycommoninbacteria,butisapparentlyabsentfromarchaea
pend on substrate-level phosphorylation only (members of the andeukaryotes.Interestingly,genomicanalysesrevealthatPTS
ordersThermococcalesandDesulphurococcales)[12].However, is also lacking from the thermophilic bacteria Thermotoga
comparative genomic analyses of Thermococcales (e.g. Pyro- maritimaandAquifexaeolicus.Transportedoligosaccharidesare
coccus spp.) have revealed the presence of putative membrane- hydrolysedfurthertoglucosebyspecificintracellularglycoside
potential-generating protein complexes [15,16] that require hydrolases[19,24,29,36–39].Glucoseismetabolizedtopyruvate
experimentalverification. viavariantsoftwomainsugarcatabolicroutes:theEMpathway,
ThesaccharolyticarchaeaPyrococcusfuriosusandSulfolobus thatisoperatinginPyrococcusfuriosus,ortheEDpathway,which
solfataricus have been studied in considerable detail, and, as isfoundinS.solfataricus[5,13].Subsequently,pyruvateiscon-
such, form the basis of the present review. Pyrococcus furiosus verted into acetyl-CoA and CO by pyruvate ferredoxin oxido-
2
is an anaerobic micro-organism with an optimum temperature reductase[40].S.solfataricusandotherrespiringarchaeahavethe
for growth (T ) of 100◦C that was isolated from marine hot capacitytocompletelyoxidizeacetyl-CoAtoCO viathecitric
opt 2
springs,andbelongstotheEuryarchaeota[17].S.solfataricusis acid cycle, when in combination with respiratory-coupled oxi-
anaerobicmicro-organism(T =80◦C)thatwasisolatedfrom dation of NAD(P)H and ferredoxin [41]. Based on analysis of
opt
acidic solfatara fields, and belongs to the Crenarchaeota [18]. theS.solfataricusgenome,ithasbeenproposedthatferredoxin,
Both archaea are able to grow on a variety of α- and β-linked ratherthanNADH,maybethemainphysiologicalelectroncarrier
glucosesaccharidesandglucose[17,19–24].Polysaccharidesare inSulfolobus,andmaybeinarchaeaingeneral[42].Obligatory
(cid:1)c 2003Biochemical Society
234 C.H.Verheesandothers
Table2 Molecularreconstructionofarchaealandthermophilicsugarmetabolism
Numberingofthegenesisaccordingtohttp://www-archbac.u-psud.fr/projects/sulfolobus/.The16archaeaforwhichgenomesequencesareavailablehavebeenclustered(basedonsimilar
genomicprofiles)forthesakeofclarity;thegenesofthespeciesbetweenparentheseshavenotbeenincluded.PF,Pyrococcusfuriosus(PH,Pyrococcushorikoshii;PAB,Pyrococcusabyssi);MA,
Methanosarcinaacetivorans(MM,Methanosarcinamazei);MJ,Methanocaldococcusjannaschii;MT,Methanobacteriumthermoautotrophicum(MK,Methanopyruskandleri);AFU,Archaeoglobus
fulgidus;VNG,HalobacteriumNRC-1;TA,Thermoplasmaacidophilum(TV,Thermoplasmavolcanium);SSO,Sulfolobussolfataricus(STO,Sulfolobustokadaii);APE,Aeropyrumpernix;PAE,
Pyrobaculumaerophilum.ThehyperthermophilicbacteriumThermotogamaritima(TM)isincludedtoallowcomparisonofthearchaealgenomeswithabacterialgenome.Experimentallyconfirmed
geneproductsareinboldandareunderlined[46,51,52,57,58,66,67,72–74,76,81,85,86,97,98,131,132].Itshouldbestressedthattheproposedgenefunctionalityrequiresexperimentalverification;
parenthesesindicateuncertainsubstratespecificity.ThenumbersandenzymeabbreviationsareasinScheme1;EC,EnzymeCommission(http://www.genome.ad.jp/kegg);COG,Clustersof
OrthologousGroups(http://www.ncbi.nlm.nih.gov/COG).Asterisks(*)intheCOGcolumnindicateidentity;inthespeciescolumns,asterisksindicateclusteringofgenes(percolumn,genesmarked
withone,twoorthreeasteriskslinkwithothergenesmarkedsimilarly),and#indicatesafusionoftwoenzyme-encodinggenes.FortheDomaincolumn:A,Archaea;B,Bacteria;E,Eukaryota.
Euyarchaea Crenarchaea Bacteria
Number Enzyme Remark EC COG Domain PF(PH/PAB) MJ MA(MM) MT(MK) AFU VNG TA(TV) SSO(STO) APE PAE TM
EMpathway
1 GLK ADP/glucose 2.7.1.147 4809* A 0312 (1604)
ATP/glucose 2.7.1.2 0837 BE 2629 0825 2091 3437 1469
ATP/hexose 2.7.1.1 E
2 PGI 5.3.1.9 2140 A 0196 0821 (1494)
5.3.1.9 0166 ABE 1605* 1992 (1419) (2281) [phone]
3 PFK ADP 2.7.1.146 4809* A 1784 1604* 3562
ATP 2.7.1.11 0205 ABE 0012* 0835 0209
PP 2.7.1.90 ABE 0289 0289
i
4 FBA 4.1.2.13 1830 A 1956 0400 0439 0579 0108 0683 3226 0011*
4.1.2.13 0191 BE 0273
5 TIM 5.3.1.1 0149 ABE 19204 1528 4607 1041* 1304 1027 0313 2592 1538 1501 0689*#
6/7 GAPOR Ferredoxin 1.2.1.- 2414 A 0464 1185 1029
GAPN NAD 1.2.1.- 1012 A (0755) (1411) (1355) [phone] 3194*3 1786
6 GAPDH NAD(P) 1.2.1.12 0057 ABE 18744 1146 1018 1009 1732 0095 1103 0528** 0171** 1740* 0688*
7 PGK ADP 2.7.2.3 0126 ABE 1057 0641 2669 1042* 1146 1216 1075 0527** 0173** 1742* 0689*#
8 PGM 2,3-PG 5.4.2.1 0406 ABE 1347 2236 1374
Independent 5.4.2.1 0696 ABE 1959 1612 0132 1591 1751 1887 0413 0417 1616 2326 1774
9 Enolase 4.2.1.11 0148 ABE 0215 0232 1672 0043 1132 1142 0882 09135 2458 0812 0877
10 PYK ADP 2.7.1.40 0469 ABE 11882 0108 3890 0324 0896 09815 0489 0819 0208
Gluconeogenesis
21 PPS ATP 2.7.9.2 0574 ABE 0043 0542 2667 1119 0710 0330 0886 0883 0650 2423 0272
9 Enolase 4.2.1.11 0148 ABE 0215 0232 1672 0043 1132 1142 0882 0913 2458 0812 0877
8 PGM 2,3-PG 5.4.2.1 0406 ABE 1347 2236 1374
Independent 5.4.2.1 0696 ABE 1959 1612 0132 1591 1751 1887 0413 0417 1616 2326 1774
7 PGK ATP 2.7.2.3 0126 ABE 1057 0641 2669 1042 1146 1216 1075 0527 0173 1742 0689
6 GAPDH NAD(P)H 1.2.1.12 0057 ABE 18744 1146 1018 1009 1732 0095 1103 0528 0171 1740 0688
5 TIM 5.3.1.1 0149 ABE 19204 1528 4607 1041* 1304 1027 0313 2592 1538 1501 0689**
4 FBA 4.1.2.13 1830 A 1956 0400 0439 0579 0108 0683 3226 0011
4.1.2.13 0191 BE 0273
22 FBP TypeV 3.1.3.11 1980 A 06131 0299 1686 1442 1428 0286 1109 0944
TypeIV 3.1.3.11 0483 ABE 2014 0109 3344 0871 2372 2418 1798 1415
TypeI 3.1.3.11 0158 ABE 0684
2 PGI 5.3.1.9 2140 A 0196 0821 (1494)
5.3.1.9 0166 ABE 1605 1992 (1419) (2281) [phone]
EDpathway
11 GlcDH NAD(P) 1.1.1.47 1063 ABE 0446* 0897 30033042**(3204) 0298
Glc-lac 3.1.1.17 3386 ABE 0648 3041**
12 G-hydr 4.2.1.39 4948** A 0442* 0085 3198*3 (0006)
13 KDGald 4.1.2.- 0329*** A 0619 3197*3
14 AldDH NAD(P) 1.2.1.- 1012 A (0809) (1629)
15 Glykin ATP 2.7.1.- 2379 A 0453 0666
16 KDGkin 2.7.1.45 0524 AB 0158 3195* 0067**
18 G6PDH NAD(P) 1.1.1.49 0364 BE 1155***
6PG-lac 3.1.1.31 0363 BE 1154***
19 GlcA-kin ATP 2.7.1.31 BE 0443
20 PG-hydr 4.2.1.12 4948** BE (0006)
17 KDPGald 0329*** A 0444*
KDPGald 4.1.2.14 0800 BE 0066**
6 GAPDH NAD(P) 1.2.1.12 0057 ABE 1874 1146 1018 1009 1732 0095 1103 0528** 0171* 1740* 0688*
7 PGK ADP 2.7.3.2 0126 ABE 1057 0641 2669 1042 1146 1216 1075 0527** 0173* 1742* 0689*
6/7 GAPOR Ferredoxin 1.2.1.- 2414 A 0464 1185 1029
GAPN NAD 1.2.1.- 1012 A (0755) (1411) (1355) [phone] 3194*3 1786
8 PGM 2,3-PG 5.4.2.1 0406 ABE 1347 2236 1374
Independent 5.4.2.1 0696 ABE 1959 1612 0132 1591 1751 1887 0413 0417 1616 2326 1774
9 Enolase 4.2.1.11 0148 ABE 0215 0232 1672 0043 1132 1142 0882 0913 2458 0812 0877
10 PYK ADP 2.7.1.40 0469 ABE 1188 0108 3890 0324 0896 0981 0489 0819 0208
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GlycolysisinArchaea 235
Table2 (Contd.)
Euyarchaea Crenarchaea Bacteria
Number Enzyme Remark EC COG Domain PF(PH/PAB) MJ MA(MM) MT(MK) AFU VNG TA(TV) SSO(STO) APE PAE TM
PPpathway
21 PGDHDC 1.1.1.44 0362 BE (0716) (2553) (1560) (1145) 0438
R5Piso 5.3.1.6 0120 A 1258 1603 1683 0608 0943 2272 0878 0978 0665 1027
5.3.1.6 0698 BE 1080
22 RPepi 5.1.3.1 0036 0680* 1718
23 Tr.ketolase N-terminalsubunit 2.2.1.1 3959* 1688* 0679* 0617* 0299* 0583* 1929* 0954*
C-terminalsubunit 3958* 1689* 0681* 0618* 0297* 0586* 1927* 0953*
Tr.aldolase 2.2.1.2 0176 0960 0616* 0295
1CharacterizedinThermococcuskodakaraensis[99].
2CharacterizedinPyrococcusfuriosus(J.E.TuiningaandS.W.M.Kengen,unpublishedresults).
3CharacterizedinSulfolobussolfataricus(B.Siebers,T.J.G.EttemaandJ.vanderOost,unpublishedresults).
4CharacterizedinPyrococcuswoesei[64,133].
5CharacterizedinThermoproteustenax[114].
anaerobic fermenting archaea such as Pyrococcus furiosus pro- GLK(Glucokinase)
duce mainly acetate and CO as end products via an archaeal-
2 The enzyme that is responsible for the activation of glucose in
specificacetyl-CoAsynthetase[43,44].
Pyrococcus furiosus is an ADP-dependent kinase (ADP-GLK)
that is not related to the classical ATP-dependent enzymes of
bacteria (GLK) or eukaryotes (hexokinase) [8,46]. The Pyro-
ARCHAEALVARIANTSOFTHEEMPATHWAY coccus furiosus GLK is member of a unique family that also
includesADP-dependentphosphofructokinases(ADP-PFKs),as
The classical EM pathway is well-conserved in bacteria and
discussed below [8,46,47]. ADP-GLK is also found in other
eukaryotes,wheretenenzymescatalysethestep-wiseoxidation
PyrococcusandThermococcusspecies[47].TheactivityofADP-
ofglucosetopyruvate(Table2).Glycolysisstartswiththephos-
GLK has been measured in extracts of Archaeoglobus fulgidus
phorylationofglucose,followedbytheisomerizationtofructose
strain 7324 [48], but the genome sequence of a closely
6-phosphate and a second phosphorylation, the aldol cleavage
related strain (VC16) does not contain a homologue of either
of fructose 1,6-bisphosphate and a P-dependent activation of
i Pyrococcus furiosus GLK or classical bacterial/eukaryal kinase
glyceraldehyde 3-phosphate (GAP), which is further converted
(Table 2). Rather than ADP-GLK, homologues of ATP-GLK
intopyruvate[1](Scheme1B).ThenetreactionofthetotalEM
are found among other archaea (Table 2), including Thermo-
pathwayis:
proteustenax,Thermoplasmavolcanium,Thermoplasmaacido-
philum,AeropyrumpernixandPyrobaculumaerophilum(www.
Glucose+2ADP+2P +2NAD+→ genome.ad.jp/kegg/kegg2.html). So far, only the archaeal ATP-
i
GLKsfromAe.pernix[49]andThermoproteustenax[50]have
2pyruvate+2ATP+2NADH+2H+
been characterized biochemically. They actually appear to
havebroadsubstratespecificity,and,assuch,theywerereferred
The best-studied archaeal EM pathway is the one of Pyro- toashexokinases.
coccusfuriosus.Biochemicalstudiesandcomparativegenomics
have revealed that only four of the ten glycolysis enzymes of
bacteria/eukaryoteshaveorthologuesinPyrococcus:triosephos-
PGI(Phosphoglucoseisomerase)
phateisomerase(TIM),phosphoglyceratemutase(PGM),enolase
and pyruvate kinase (PYK) [9] (Table 2). Novel enzymes, ThegenesencodingthePGIofPyrococcusfuriosusandThermo-
unprecedentedconversionsanddifferentcontrolpointshavebeen coccuslitoralishavebeenclonedandcharacterized[51–53].This
elucidatedinthepyrococcalglycolysis.Eachglycolyticenzyme type of PGI is unrelated to its bacterial and eukaryal counter-
is described separately below, and the case of Pyrococcus is parts.Insteaditbelongstotheso-calledcupinsuperfamily[52].
compared with that of bacteria/eukaryotes and, when data are Membersofthissuperfamilyhaveawidevarietyoffunctionsthat
available,withthatofotherarchaea.Obviously,severalglycolytic oftenappeartoberelatedtothebindingorconversionofsacchar-
enzymesareabsentinorganisms(e.g.S.solfataricus)thatdegrade ides.TheclosestfunctionalanalogueofthearchaealPGIisphos-
glucoseviaanED-likepathway(seebelow;Table2).Although phomannose isomerase [52,53a]. This type of PGI seems to be
an alternative has been proposed (see discussion below; [45]), restrictedtothePyrococcus,ThermococcusandMethanosarcina
the general view of the net reaction of the EM-like pathway of species;ageneinAr.fulgidusappearstoencodeanenzymethat
Pyrococcusfuriosusis: is more closely related to cupin proteins with distinct substrate
specificity (Table 2) [52]. The pgi gene of Pyrococcus furiosus
overlaps 25nt with an adjacent ORF (open reading frame)
Glucose+4fdox→2pyruvate+4fdred+4H+
(PF0196),suggestingthatthetwoORFsareco-transcribed.How-
ever,itwasdemonstratedbyNorthernblottingthatPGIismono-
wherefdisthesingle-electroncarrierferredoxin,whichiseither cistronic [52], a conclusion supported by microarray analysis
oxidized(fdox)orreduced(fdred). [54].
(cid:1)c 2003Biochemical Society
236 C.H.Verheesandothers
encoding ADP-dependent kinases with an unknown specificity
(Figure1).Nohomologueshavebeenfoundinbacterialgenomes.
Interestingly, however, uncharacterized genes with significant
identitywiththeADP-dependentarchaealkinaseswereidentified
in several eukaryal genomes, including that of man (Figure 1)
[57].TheADP-dependentsugarkinasesdonotshareoverallse-
quencesimilaritywithclassicalsugarkinasesequencesinbacteria
and eukaryotes [57]. The recently solved structure of ADP-
GLK from the archaeon Thermococcus litoralis, closely related
to Pyrococcus furiosus, shows a similar fold as ribokinase and
adenosine kinase [59]. The ‘minor PFK’ from Escherichia coli
(PFKB) belongs to this ribokinase family as well. In bacteria/
eukaryotes,theATP-dependenthexokinases/GLKsononehand,
and the ATP-dependent PFKs on the other, belong to different
monophyleticfamilies(COG0837and0205respectively).Hence,
based on the established primary and the tertiary structures, it
is concluded that the archaeal ADP-dependent kinases are not
Figure1 ADP-dependentkinasesinarchaeaandeukaryotes relatedtotheircounterpartsinbacteriaandeukaryotes.
The genome sequences of some hyperthermophilic archaea
PhylogeneticanalysisofADP-dependentkinasesinarchaea(PF,Pyrococcusfuriosus;PH,
suchasPyrobaculumaerophilumandAe.pernixcontainhomo-
Pyrococcushorikoshii;PAB,Pyrococcusabyssi;MJ,Methanocaldococcusjannaschii;MM,
logues of bacterial/eukaryal ATP-dependent sugar kinases. The
Methanosarcinamazei;MA,Methanosarcinaacetivorans).Redbranches,PFKs;bluebranches,
GLKs; also included are related kinases (green branch) with unknown specificity from PFKofAe.pernixhasbeencharacterizedandrepresentsthefirst
Methanosarcinaspp.(MM0472,MA3562)andHomosapiens(Man;GeneIdentifier13543940). ATP-dependent PFK to be characterized from a hyperthermo-
ThedendrogramwasbasedonaClustalXalignment. phile, an enzyme that is apparently not allosterically regulated
[60]. A third type of PFK exists in Thermoproteus tenax. This
enzymeisPP-dependentandisalsofoundamongsomebacteria
i
and eukaryotes, and is distantly related to ATP-PFK [61] (both
GenomeanalysisrevealsthatorthologuesofclassicalPGIare belongingtoCOG0205)(Table2).ThegenomeofAr.fulgidus
presentnotonlyinthebacteriumThermotogamaritima,butalsoin strain VC16 appears not to encode any homologue of PFK
several archaea, such as Methanocaldococcus (Methanococcus) (Table2),althoughincellextractsofstrain7423,ADP-PFKacti-
jannaschii and Halobacterium NRC-1 (Table 2). According to vityhasbeendetected[48].
the COG (clusters of orthologous groups of proteins) database
(http://www.ncbi.nlm.nih.gov/COG), additional orthologues are
presentinthegenomesofThermoplasmaacidophilum(TA1419),
S.solfataricus(SSO2281)andAe.pernix(APE0768).However, FBA(Fructose-1,6-bisphosphatealdolase)
only a sugar isomerase (SIS) domain is identified in the latter A distantly related archaeal type of FBA has recently been
protein sequences, whereas both a SIS domain and a phospho- identifiedinThermoproteustenaxandPyrococcusfuriosus[62],
glucose(PGI)domainarepresentintheotherorthologues.Apart confirming earlier function prediction [63]. Orthologues of this
from PGIs, the SIS domain has also been demonstrated to play ‘archaeal type class I aldolase’ are present in all sequenced
a role in the isomerization of other sugars [55]. Thus TA1419 archaeal genomes, except for Thermoplasma and Pyrobaculum.
and APE0768 cannot unambiguously be classified as PGIs Paralogues of the aldolase are present in Methanocaldococcus
(Table2). jannaschii(MJ1585),Ar.fulgidus(AF0230),andHalobacterium
NRC-1 (VNG0309), and the encoded enzymes were predicted
tofunctioneitherasdeoxyribosephosphatealdolaseorastrans-
aldolase[63].
PFK
Analysis of PFK from Pyrococcus furiosus again revealed that
this kinase is ADP-dependent, just like its counterpart from
TIM
the related Thermococcus zilligii [4,46,56]. Sequence analysis
indicatedthattheADP-PFKsareparaloguesoftheADP-GLKs, AllsequencedarchaeahaveanobviousTIMhomologue(Table2),
sharingthesameCOG4809(Table2andFigure1).Anorthologue indicating that TIM does not display extensive variation, unlike
of the Pyrococcus furiosus ADP-PFK was also identified in someoftheotherglycolyticenzymes.Althoughthisenzymedoes
thenon-saccharolyticMethanocaldococcusjannaschii[57].The nottakepartintheEDpathway(regularormodified),itseemsto
characterized enzyme (MJ1604) has recently been reported to berequiredforthoseorganismsharbouringanED-typepathway
have dual activity towards both glucose and fructose 6-phos- apparentlyinordertoperformgluconeogenesis.Ithaspreviously
phate [58]. ADP-PFK homologues appeared to be present in been noticed that TIM of hyperthermophiles is smaller (220–
the sulphate-reducing Ar. fulgidus strain 7324 [48], and in both 230aminoacids)thanTIMfoundinmesophilessuchasE.coli
thermophilic and mesophilic glycogen-degrading methanogenic (250–260 amino acids) [64]. The structure of the enzyme from
species belonging to the Methanococcales and the Methanosar- the hyperthermophile Pyrococcus woesei has been determined
cinales[57].ThepfkgeneofMethanocaldococcusjannaschiiis andcomparedwiththatofthemesophilichomologue.Itisamore
located adjacent to predicted homologues of glucose isomerase compactprotein(‘tinyTIM’)[65],withseveralpeptides‘missing’
(MJ1605) as well as glycogen synthase (MJ1606) (Table 2), throughouttheproteinsequence.Interestingly,allarchaeapossess
stronglysuggestingaconcertedroleinglycogensynthesis.Inter- thissmallerversionofTIM,whereaseukaryotesandbacteria,as
estingly,thegenesencodingthepredictedADP-PFKsinMethano- wellasthehyperthermophilicbacteriumAq.aeolicus,containthe
sarcina spp. are clustered with paralogous genes, probably largerversion.
(cid:1)c 2003Biochemical Society
GlycolysisinArchaea 237
GAPOR(Glyceraldehyde-3-phosphateferredoxinoxidoreductase) (Table2).IthasbeendemonstratedthatbothMJ0010andMJ1612
encodeenzymeswithPGMactivity[73].Thephysiologicalroleof
In bacteria and eukaryotes, the (reversible) conversion of GAP
these paralogues remains to be addressed. This new subfamily
into3-phosphoglycerateiscatalysedbytheenzymecoupleofthe
of PGM is present in all archaea whose genome has been se-
NAD+-dependent GAPDH (glyceraldehyde-3-phosphate dehy-
quenced(Table2).Asinseveralbacteria,twoPGMtypescoexist
drogenase) (GAPN) and the ATP-generating phosphoglycerate
inSulfolobusandThermoplasma[72];thisapparentredundancy
kinase (PGK). In Pyrococcus furiosus, a distinct enzyme is
would makesensewhenbothsystemsevolveddistinct catalytic
responsibleforthesingle-stepconversionofGAPinto3-phospho-
features.
glycerate in a phosphate-independent manner: GAPOR [66].
Unlike the bacterial system, the physiological role of GAPOR
appears to be solely the oxidation of GAP (glycolysis) and not
Enolase
thereductionof3-phosphoglycerate[67](seetheRegulationof
archaealglycolysissection,below).GAPORisamemberofthe The enolase of Pyrococcus furiosus has been characterized and
aldehyde oxidoreductase (AOR) family of tungsten-containing shows high similarity to enolases from various sources [74].
enzymes.ThreeofthefiveAOR-familyparaloguesofPyrococcus Enolasescanbeidentifiedinallarchaealgenomesandrepresent
furiosus [AOR, FOR (formaldehyde ferredoxin oxidoreductase) thebacterial/eukaryaltype.Theyappeartobehighlyconserved
and GAPOR] have been characterized biochemically, and each throughoutthethreedomainsandhavebeenhelpfulinunravelling
catalyses the ferredoxin-dependent oxidation of aldehydes [68]. phylogeneticrelationships[75].
The crystal structure of AOR revealed that the tungsten is co-
ordinatedbytwopterinmolecules,andthattheproteincontains
a[4Fe–4S]cluster[68].Thepterinligandsandtheiron–sulphur PYK
clusterareco-ordinatedbyconservedaminoacidresidues.Based
As in bacteria and eukaryotes, the final step in the archaeal
on sequence alignments, a typical GAPOR signature in the
glycolyticpathwaysappearstobecatalysedbyPYK.Homologues
[4Fe–4S]cluster-co-ordinatingresidueswasobserved(Cys-Gly-
ofPYKarepresentinthegenomesofsaccharolyticarchaea,and
Glu-Pro-Cys-Pro-Xaa-Xaa-Cys), while the other AOR family
appear to be absent in the autotrophs Methanopyrus kandleri
members contain a less conserved signature (Cys-Xaa-Xaa-
and Methanobacterium thermoautotrophicus, as well as in the
Cys-Xaa-Xaa-Xaa-Cys) [67]. Using this conserved signature,
non-saccharolyticAr.fulgidusstrainVC16(Table2).Ithadbeen
potential homologues of GAPOR can be found in Pyrococcus
suggestedthatthePyrococcusfuriosusPEPsynthase(PPS)might
horikoshii, Pyrococcus abyssi, Pyrobaculum aerophilum and
play a novel glycolytic role, synthesizing ATP from PEP and
Methanocaldococcusjannaschii(Table2).GAPORactivitywas
AMP [45], but kinetic data do not support such a conclusion
measured in cell extracts of Methanocaldococcus jannaschii
[76].
(G. J. Schut and J. van der Oost, unpublished work), the
Enzyme-activity measurements of key glycolytic enzymes in
starch-degrading Ar. fulgidus strain 7324 [48], Desulfurococcus
various archaea on the one hand, and comparative genomics on
amylolyticus and Thermococcus litoralis [5]. The presence of
the other (Table 2), indicate that archaeal EM pathways con-
GAPORinMethanocaldococcusjannaschiiprobablyreflectsthe
stitutedifferentcombinationsofclassical(bacterial/eukaryal)and
capacityofthismethanogentodegradeglycogen[57].
novel (archaeal) enzymes. For example, D. amylolyticus was
An interesting variation occurs in the crenarchaeon Thermo-
found to contain a partially modified EM pathway, including
proteustenax,whichcontainsadistinctallostericallycontrolled
GAPOR,butwithclassicalATP-GLKandATP-PFK(Scheme2)
GAPN that catalyses the phosphate-independent, single-step
[5,6,60].TheThermoproteustenaxEMpathwayincludesanATP-
oxidation of GAP to 3-phosphoglycerate in glycolysis [69].
GLK,aPP-PFKandGAPN(Scheme2)[5,61].Itisconcluded
AlthoughbothGAPORandGAPNarenovelglycolyticenzymes i
that all archaeal EM pathways that have been characterized to
only identified in archaea, they both have distantly related
date are relatively inefficient: during the oxidation of glucose
enzymes, at least in bacteria. In enterobacteria, such as E. coli,
to pyruvate by Pyrococcus furiosus, no net ATP appears to be
a molybdenum-containing oxidoreductase has been identified
produced(Scheme1A).Thisisadirectconsequenceofthesingle-
that is structurally related to the AOR family, but is certainly
step, non-phosphorylating conversion of GAP by GAPOR or
not involved in glycolysis [67]. GAPN is member of a large
GAPN,incontrastwithsubstrate-levelphosphorylationbytheen-
family of aldehyde dehydrogenases that are also present in
zymecoupleGAPDH/PGKinclassicalglycolysis(Scheme1B).
bacteria and eukaryotes [69]. The physiological role of the
The ADP-dependent sugar kinases (Pyrococcus furiosus) do
numerous GAPOR/AOR and GAPN-like enzymes that appear
not appear to affect the overall glycolytic efficiency. The PP-
tobeencodedbymanyarchaealgenomes(Table2)remainstobe i
PFK(Thermoproteustenax)appearstopartiallyrestoretheloss
established.
of efficiency, since a waste product (PP) is used as phos-
i
phoryl donor; however, the yield of the Thermoproteus tenax
pathway (1ATP/glucose) would still be less than in classical
PGM
glycolysis (2ATP/glucose). Another difference is that GAPOR
The last ‘missing-link’ of the Pyrococcus furiosus glycolytic activityinThermococcus,Pyrococcus,Desulfurococcusspp.and
pathway was PGM, responsible for interconverting 3-phospho- Ar. fulgidus strain 7324 results in the production of reduced
glycerate and 2-phosphoglycerate. Archaeal PGM has been ferredoxin,insteadofNADHinclassicalglycolysis.Althoughthe
predicted by comparative analysis of metabolic pathways in abovediscussionsuggeststhatthePyrococcusfuriosusglycolysis
different genomes [9,70,71]. The prediction of the archaeal doesnotresultinthenetgenerationofATP,ithasbeendeduced
PGMs (11% amino acid identity with its E. coli counterpart) fromyieldstudiesthatPyrococcusfuriosushasanetproduction
hasbeenconfirmedexperimentallyforPyrococcusfuriosusand ofatleast1molofATPduringtheglycolyticoxidationof1molof
Methanocaldococcus jannaschii (MJ1612) [72]. Apparently, a glucosetopyruvate[4].Thiscouldbeachievedbythefunction
gene-duplication event has led to a second copy of this gene in ofPPSinglycolysis(PEP+AMP+P →pyruvate+ATP)[45],
i
Methanocaldococcus jannaschii (MJ0010), Methanobacterium orbyanalternativemembrane-potential-generatingsysteminvol-
thermoautotrophicus (MT0418) and Ar. fulgidus (AF1425) vingferredoxinandhydrogenase[15].
(cid:1)c 2003Biochemical Society
238 C.H.Verheesandothers
Scheme2 Overviewofglucosemetabolisminarchaea
Summaryofrelevantvariationsintheglucose-degradingpathwaysofarchaea,ascomparedwiththeclassicalbacterial/eukaryalpathways.Pyr:FdOR,pyruvateferredoxinoxidoreductase;TCA,
trichloroaceticacid.AdaptedfromArchivesofMicrobiology,ComparativeanalysisoftheEmbden–MeyerhofandEntner–Doudoroffglycolyticpathwaysinhyperthermophilicarchaeaandthebacterium
Thermologa,Selig,M.,Xavier,K.B.,Santos,H.andScho¨nheit,P.,167,p.230,Figure11,1997(cid:1)c Springer-VerlagGmbH&Co.[5].
ED-LIKEPATHWAYSINARCHAEA bysubstrate-levelphosphorylationduringconversionofGAPvia
1,3-diphosphoglycerateinto3-phosphoglycerateinthecanonical
The classical ED pathway of bacteria (and a limited number
EMpathway(Scheme1B)[11].Thermoproteus,Thermoplasma
of eukaryal micro-organisms) consists of nine enzymes, and
andSulfolobusspp.useanED-likepathwayinwhichnoneofthe
starts with the phosphorylation of either glucose or its oxidized
hexoseintermediatesarephosphorylated.Instead,theactivation
derivativegluconate(Scheme1B).Thegenerated2-keto-3-deoxy-
via phosphorylation does occur at a later stage in the pathway:
6-phosphogluconate(KDPG)isconvertedbyanaldolcleavage,
at the level of KDG (e.g. halophilic archaea) or glycerate (e.g.
resulting in the formation of GAP and pyruvate. The triose
Sulfolobus and Thermoplasma) (Scheme 1). Whereas the ED
phosphateisactivatedbyP,andisfurtherdegradedtopyruvate
i pathwayinhalophiles(viaKDPG)isexpectedtohaveasimilarnet
byenzymesthatareidenticalwiththoseoftheEMpathway[1].
resultasthebacterialroute,thereappearstobenonetgenerationof
ThenetreactionofthebacterialEDpathwayis:
ATPbythelatter‘non-phosphorylating’EDpathway(viaKDG)
(Scheme1A):
Glucose+ADP+P +NAD++NADP+→
i
2pyruvate+ATP+NADH+NADPH+2H+ Glucose+2NAD(P)+→2pyruvate+2NAD(P)H+2H+
It should be noted that the main difference between the ED Both archaeal ED variants merge at some stage with the en-
and EM pathways is that, in the former, both NADPH and zymesoftheC -stageoftheEMpathway[5,11,77–79].Specific
3
NADHareformed,insteadof2NADH,andthatsubstrate-level featuresofthearchaealEDpathwayarediscussedbelow.
phosphorylation only yields a single ATP per glucose molecule
[1].
Glucosedehydrogenase
ED-likepathwaysfoundinarchaeaaremodifiedintwoways.
Halophilicarchaea(andsomebacteria)useavariantEDpathway The first enzyme of the variant ED pathways of archaea is the
in which 2-keto-3-deoxygluconate (KDG) is phosphorylated to NAD(P)+-dependentglucosedehydrogenase[80].Thefirstgene
KDPG by KDG kinase, rather than the general conversion of encoding an archaeal glucose dehydrogenase was isolated from
glucoseintoglucose6-phosphateinbacteriaandsomeeukaryotes Thermoplasmaacidophilum[81].Subsequentlyhomologueswere
(Scheme 1A). Phosphorylation at either level of the C -stage in purifiedfromcellextractsofThermoproteustenaxandHaloferax
6
thepathway(glucose,gluconateorKDG)allowsthegainofATP mediterranei[82,83].Threeparaloguesofthisproteinsequence
(cid:1)c 2003Biochemical Society
GlycolysisinArchaea 239
wereidentifiedinthegenomeofS.solfataricus,andasinglecopy Glyceratekinase
wasidentifiedinthegenomeofHalobacteriumNRC-1(Table2).
Phosphorylation activity on glycerate has been demonstrated in
The protein sequences of these archaeal glucose dehydroge-
Thermoplasma acidophilum cell extracts. The absence of PGM
nasesresemblezinc-containingalcoholdehydrogenases,andare
activitysuggeststhat2-phosphoglycerate(insteadof3-phospho-
distantly related to bacterial/eukaryal glucose dehydrogenases
glycerate)isproducedfromthephosphorylationreactionofgly-
(COG1063;Table2).
cerate[79].Basedonthesedata,thisconversionhasbeenproposed
tooccurinSulfolobusaswell[42,87].Candidategenesthatmight
encode glycerate kinase have been identified in the genome of
S.solfataricus[42],andinthatofotherarchaea(Table2).PGM
Gluconatedehydratase
proteinsequences(dPGM-type;Table2)thathavebeenidentified
A potential gluconate dehydratase (SSO3198) was identified in in S. solfataricus and Thermoplasma acidophilum would pro-
thegenomeofS.solfataricusbasedonthepresenceofadehydra- bably be produced only under gluconeogenic conditions. The
tase-like domain, and on its clustering with KDG aldolase specificityoftheproposedglyceratekinasesandthePGMsremain
(SSO3197; see below), a kinase (SSO3195) and a GAPN toberesolved.
homologue (SSO3194) (Table 2) (T. J. G. Ettema and J. van Overall, the ED pathways of Sulfolobus and Thermoproteus
derOost,unpublishedwork).Clusteringofthesegenescouldnot yield:glucose→2pyuvate+2NADPH,duetotheapparentlack
be observed in any of the other sequenced archaeal genomes. of a GAP oxidation/phosphorylation system. Therefore, it does
AnorthologueofSSO3198wasidentifiedinthegenomeofthe notmatteratwhatlevelphosphorylationoccursinthesearchaea.
bacteriumThermotogamaritima(TM0006),whereitmayencode SincethearchaeathatcontainEDpathwaysappeartobecapable
themissingphosphogluconatedehydratase(Table2). ofarelativelyefficientcofactorreoxidation,eitherviaoxidative
phosphorylation(e.g.Sulfolobus)orviasulphurrespiration(e.g.
Thermoproteus),thelowefficiencyoftheirEDpathwaymightbe
of less importance. Alternatively, it has been suggested that the
KDGkinase
archaealnon-phosphorylatingEDpathwaymaybereversible,i.e.
Strains of the halophile genera Halobacterium, Haloferax and analternativegluconeogenesispathway(reviewedin[1]).
HalococcusappeartobetheonlyarchaeathatphosphorylateC
6
metabolites of the ED-like pathway via the phosphorylation of
KDG by a specific kinase (Scheme 1). The generated KDPG PENTOSECONVERSIONINARCHAEA
appearstobeconvertedasinthebacterialEDpathway(reviewed
Somearchaea(e.g.Sulfolobus)havethecapacitytousepentoses
in[84]),and,assuch,istheonlyexampletodateofanarchaeal
as a carbon source. Moreover, all autotrophic and heterotrophic
ED pathway with net ATP production (however, see discussion
archaeamustbeabletogeneratepentosesasbuildingblocksfor
below).
the biosynthesis of nucleotides and certain amino acids. Hence
all archaea should possess enzymes that are responsible for the
pentose metabolism. At present, however, little information is
availableontheidentityofthearchaealenzymesorontheactual
KDGaldolase
compositionofthepathway(s)involved.
A novel aldolase that catalyses the conversion of the non- A general pathway for pentose degradation and biosynthesis
phosphorylated KDG has been purified and characterized from in bacteria and eukaryotes is the PP pathway, consisting of
S.solfataricus[85].InthegenomeofHalobacterium,adistantly an oxidative and a non-oxidative branch [1]. Obviously, the
relatedgene(VNG0444)hasbeenidentifiedbysimilaritysearch non-oxidative branch is essential for growth on pentoses. In
andgenecontext,andwaspredictedtoencodetheKDPGaldolase addition, there may be a link with the gluconate metabolism
(Table 2). Recently, biochemical evidence has been gained that (Scheme 1B). The oxidative branch is used to produce C
5
TM0066 in the Thermotoga maritima genome (orthologous to sugars (ribulose 5-phosphate, xylulose 5-phosphate and ribose
VNG0444)indeedencodesanactiveKDPGaldolase[86]. 5-phosphate)fromC sugars(6-phosphogluconate)bymeansof
6
oxidationanddecarboxylationreactions,releasingCO (Scheme
2
1B). Together with the non-oxidative branch, it forms a bypass
between glucose 6-phosphate and fructose 6-phosphate, i.e. the
Glyceraldehydedehydrogenase
hexosemonophosphatepathway.BiosyntheticfunctionsofthePP
An archaeal glyceraldehyde dehydrogenase sequence has not pathwayarethesynthesisofNADPHandribose.Atpresent,there
yet been identified unambiguously. Several GAPN homologues is no evidence that catabolism of glucose in archaea proceeds
are present in Sulfolobus, which are predicted to encode via a PP-like pathway (Scheme 1A). Genes encoding canonical
aldehydedehydrogenases[42];thespecificityoftheseparalogues enzymes of the oxidative branch of the PP pathway, such as
remainstobeestablishedexperimentally.Ithasbeenconfirmed glucose-6-phosphate dehydrogenase and gluconate-6-phosphate
biochemically that glyceraldehyde is specifically converted into dehydrogenases, have not been identified in archaea (Table 2)
glycerate by a glyceraldehyde dehydrogenasein Thermoplasma [42,88,89]. However, the possibility cannot yet be excluded
acidophilumandS.solfataricus[79,87].InHalobacteriumNRC- that the archaeal enzymes that constitute a PP-like pathway are
1, GAP is generated by aldol cleavage of KDPG, as in bacteria unrelatedoronlydistantlyrelatedtotheirclassicalcounterparts,
(Schemes1Aand1B).Althoughahomologueofanon-phosphor- andarethereforenoteasilydetectablebycomparativegenomics.
ylatingGAPOR(GAPN;VNG0937)appearstobeencodedby As pointed out above, most, if not all, archaea are expected
thegenomeofHalobacteriumNRC-1,theoxidationofGAPmay tohavethecapacitytogeneratepentoses,mostlikelyviaanon-
alsobecatalysedbytheclassicalGAPDH/PGKcouple(Table2). oxidative branch of the EM/gluconeogenesis pathway. Genome
Among the glycolytic pathways in archaea, the ED pathway analysis indicates that some of the key enzymes may indeed be
of Halobacterium is most similar to its bacterial (eukaryal) present in several archaea: transketolase, ribulose-5-phosphate
counterpart. 3-epimerase, ribulose-5-phosphate isomerase (Table 2 and
(cid:1)c 2003Biochemical Society
240 C.H.Verheesandothers
Scheme 1C). Interestingly, the recent DNA microarray analysis Specificfeaturesofthearchaealgluconeogenesispathwaywillbe
on the complete Pyrococcus furiosus genome revealed signi- discussedbelow.
ficant up-regulation on maltose (versus peptides) of the amino-
acid-biosynthesis pathways, including the biosynthesis routes
of histidine and the aromatic amino acids [90]. In the latter PPS
biosynthesis pathways, ribose 5-phosphate is an essential pre-
Homologues of classical PPS sequences are present in all
cursor.Thepredictedtransketolasegenes(PF1688andPF1689),
sequencedarchaealgenomes,despitetheirsaccharolyticcapacity,
whoseproductscatalysetheconversionoffructose6-phosphate
suggesting that the function of the enzyme in gluconeogenesis.
andGAPintoerythrose4-phosphateandthepentosexylulose5-
ThePPSfromPyrococcusfuriosuswaspurifiedandcharacterized
phosphate(Scheme1C),resideinageneclusterthatisinvolved
by two groups [45,76]. Strikingly, transcription of the gene
inthebiosynthesisofaromaticaminoacids.Indeed,thisstrongly
encodingthePPSshasbeenreportedtobeenhancedbygrowth
suggeststhatthetransketolase(togetherwithspecificisomerases)
on maltose [95], possibly suggesting a role in glycolysis (see
is involved in the synthesis of pentoses from glycolytic inter-
alsobelow).However,thelatterfindingisnotinagreementwith
mediates in archaea. It is obvious that several missing links in
recentbiochemicalandDNAmicroarrayanalyses,whichwould
thispathwaystillremaintobediscovered.Alternativesynthesis
suggest the enzyme to function in gluconeogenesis [76,90,96].
ofpentosesfrom GAP andacetaldehydehasrecentlybeensug-
In that case, classical PYK present in this archaeon (Table 2)
gestedtobecatalysedbyparaloguesoftheFBAsequencefrom
mightcatalysethereactionintheglycolyticdirection.Despitethe
Methanocaldococcus jannaschii (MJ1585) and Ar. fulgidus
bioenergeticdata,thetruefunctionofPPSinPyrococcusfuriosus
(AF0230)[63].
isstillamatterofdebate,andisundercurrentinvestigation(J.E.
Anovelglycolyticpathwaythatpotentiallyinvolvespentoses
TuiningaandS.W.M.Kengen,unpublishedwork).
has recently been proposed in Thermococcus zilligii. Based on
[13C]glucose-labelling experiments, it has been suggested that
a C6 intermediate (gluconate 6-phosphate) is cleaved into a C5 GAPDH/PGK
intermediate (xylulose 5-phosphate) and formate, involving a
All archaea contain the enzyme couple GAPDH and PGK
novel type lyase [91]. The PP is presumably degraded via the
(Table 2). It has been shown for Pyrococcus furiosus and
pentosephosphoketolasepathway,commonlyfoundinlacticacid
Thermoproteus tenax that this enzyme couple acts solely in
bacteria.[92].Arelativecontributionof2:1(novelpathwayversus
gluconeogenesis,whichalsoexplainsitspresenceinnon-sugar-
theEMpathway)wasdetectedforcellsgrownontryptone.The
degrading M. thermoautotrophicum and Ar. fulgidus strain VC-
presence of glucose in the growth medium appears to repress
16.ThenovelGAP-oxidizingenzymes,GAPORandGAPN,have
the enzymes in this novel pathway, and results in the inversion
beenshowntofunctionsolelyinglycolysisinPyrococcusfuriosus
oftherelativecontributionsofthetwopathways[91].Analter-
andThermoproteustenaxrespectively[66,67,69].
native interpretation of the labelling pattern would be the con-
version:
FBP
Fructose6-phosphate→3-oxo-6-phosphate-hexulose
AhomologueofacanonicalFBP(typeI)couldbeidentifiedinthe
→formaldehyde+ribulose5-phosphate archaealgenomesequenceofHalobacteriumNRC-1.Noobvious
orthologuesofthisgenearepresentintheotherarchaealgenomes
→formate+xylulose5-phosphate (Table2).However,characterizationofabifunctionalFBP/myo-
inositol-1-phosphatase from Methanocaldococcus jannaschii
(MJ0109)[97]resultedintheidentificationofthecorresponding
involving two isomerases, a lyase/synthase and the well-
gene in the genomes of several archaea as well as Thermotoga
characterizedFOR.Obviously,amoredetailedanalysisofenzyme
maritima(FBPtypeIV)(Table2)[98].Recentstudiesrevealed
activitiesisrequiredtoaddressthismatter.
the presence of yet another FBP (type V) in Thermococcus
kodakaraensis[99].Homologuesofthisenzymewereidentified
inall(exceptHalobacteriumspp.)euryarchaealandcrenarchaeal
GLUCONEOGENESISINARCHAEA
genomes(Table2),indicatingthatthearchaeaapparentlycontain
Gluconeogenesisisaubiquitousbiosyntheticpathwayviawhich two distinct types of FBPs, probably with functions in gluco-
pyruvateisconvertedintofructose6-phosphateorglucose6-phos- neogenesisandthePPpathway(Table2).Anorthologueofthe
phate,andsometimesallthewayintoglucose(asinmulticellular lattergeneappearstobeup-regulatedduringoperationofgluco-
eukaryotes)(Scheme1C).Severalofthepathway’sintermediates neogenesis in Pyrococcus furiosus (see below), and, as such, is
serve as precursors for a large number of anabolic pathways, themostlikelycandidateforthearchaealFBP(G.J.Schutand
including the aforementioned ribose synthesis (PP pathway). In M. W. W. Adams, unpublished work). It should be noted that
addition,severalarchaea(e.g.methanogensandSulfolobus)[93] thePP-dependentPFKofThermoproteustenaxhasbeenshown
i
havebeendemonstratedtohavethecapacitytogenerateglucose to have phosphatase activity as well [61]; it is not yet known,
polymers(glycogen)asstoragematerial.Thegluconeogenicpath- however,whetherornotthisenzymealsosubstitutesFBPinvivo.
waysharesmanyenzymeswiththeEMpathway,andistherefore ArchaeathatdonotpossessPGIproteinsequences(e.g.Meth-
partly reversible to the EM pathway. However, three enzymes anobacterium thermoautotrophicus and Ar. fulgidus) (Table 2)
are rather specific for the bacterial/eukaryal gluconeogenesis might possess a gluconeogenesis pathway up to the level of
(irreversible) and are thus not present in glycolysis: (i) the fructose 6-phosphate, which probably suffices for a link to the
phosphorylationofpyruvatetoPEPiscatalysedbyPPSinsteadof non-oxidativePPpathway,butcertainlynottoglycogensynthesis
byPYK,(ii)thedephosphorylationoffructose1,6-bisphosphate (Scheme 1C). As pointed out above, it has been suggested that
iscatalysedbyfructose-1,6-bisphosphatase(FBP)insteadofby thearchaealnon-phosphorylatingEDpathwaymaybereversible
ATP-PFK,and(iii)insomeorganisms,glucose6-phosphatecan [1]; future research should reveal whether alternative pathways
be dephosphorylated to glucose by glucose-6-phosphatase [94]. forgluconeogenesisexist.
(cid:1)c 2003Biochemical Society