Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation ChristopherBräsen,DominikEsser,BernadetteRauch,BettinaSiebers MolecularEnzymeTechnologyandBiochemistry,BiofilmCentre,FacultyofChemistry,UniversityofDuisburg-Essen,Essen,Germany SUMMARY...................................................................................................................................................91 INTRODUCTION..............................................................................................................................................91 MODIFICATIONSOFTHEEMBDEN-MEYERHOF-PARNASPATHWAYINARCHAEA.........................................................................92 GlucosePhosphorylation..................................................................................................................................94 D ADP-dependentglucokinase...........................................................................................................................94 o ROKhexokinase.........................................................................................................................................95 w Otherarchaealsugarkinases............................................................................................................................95 n PhosphoglucoseIsomerase................................................................................................................................98 lo Phosphoglucoseisomerase/phosphomannoseisomerase..............................................................................................98 a d Cupin-typephosphoglucoseisomerase...............................................................................................................100 e Phosphofructokinase.....................................................................................................................................100 d Pyrophosphate-dependentphosphofructokinase.....................................................................................................100 f r ADP-dependentphosphofructokinase................................................................................................................100 o ATP-dependentphosphofructokinase(PFK-B).........................................................................................................101 m Fructose-1,6-BisphosphateAldolase(Catabolic)..........................................................................................................101 h TriosephosphateIsomerase..............................................................................................................................102 t t Glyceraldehyde3-PhosphateOxidation..................................................................................................................103 p : Glyceraldehyde-3-phosphate:ferredoxinoxidoreductase.............................................................................................104 // m Nonphosphorylatingglyceraldehyde-3-phosphatedehydrogenase..................................................................................104 Glyceraldehyde-3-phosphatedehydrogenase(phosphorylating)(catabolic).........................................................................105 m PhosphoglycerateKinase.................................................................................................................................106 b r PhosphoglycerateMutase................................................................................................................................107 . a 2,3-Bisphosphoglyceratecofactor-dependentphosphoglyceratemutase............................................................................107 s Cofactor-independentphosphoglyceratemutase....................................................................................................107 m Enolase...................................................................................................................................................108 .o PhosphoenolpyruvateConversiontoPyruvate..........................................................................................................108 r g Pyruvatekinase........................................................................................................................................108 / MODIFICATIONSOFTHEENTNER-DOUDOROFFPATHWAYINARCHAEA................................................................................109 o n GlucoseDehydrogenase.................................................................................................................................110 GluconateDehydratase..................................................................................................................................113 D 2-Keto-3-DeoxygluconateKinase.........................................................................................................................114 e c 2-Keto-3-Deoxy-(6-Phospho)GluconateAldolase[KD(P)GA].............................................................................................114 e GAPConversion..........................................................................................................................................115 m Glyceraldehydedehydrogenase/(glycer)aldehyde:ferredoxinoxidoreductase........................................................................115 b GlycerateKinase..........................................................................................................................................116 e r GLUCONEOGENESIS........................................................................................................................................116 1 PhosphoenolpyruvateSynthetase.......................................................................................................................116 8 Pyruvate:PhosphateDikinase.............................................................................................................................117 , 2 PhosphoenolpyruvateCarboxykinase....................................................................................................................117 0 Glyceraldehyde-3-PhosphateDehydrogenase/PhosphoglycerateKinase...............................................................................119 1 BifunctionalFructose-1,6-BisphosphateAldolase/Phosphatase..........................................................................................119 8 PENTOSEDEGRADATIONPATHWAYSINARCHAEA.......................................................................................................120 b y PentoseOxidationandXyloseDehydrogenase/ArabinoseDehydrogenase.............................................................................122 g C SugarAcidDehydrationandXylonateDehydratase/ArabinonateDehydratase......................................................................125 5 u 2-Keto-3-DeoxyxylonateDehydratase/2-Keto-3-DeoxyarabinonateDehydratase.......................................................................126 e (cid:2)-KetoglutarateSemialdehydeDehydrogenase.........................................................................................................126 s t 2-Keto-3-Deoxyxylonate(KDX)/2-Keto-3-Deoxyarabinonate(KDA)Cleavage............................................................................127 ConversionofGlycolaldehydetoMalate.................................................................................................................127 (continued) AddresscorrespondencetoChristopherBräsen,[email protected], orBettinaSiebers,[email protected]. Copyright©2014,AmericanSocietyforMicrobiology.AllRightsReserved. doi:10.1128/MMBR.00041-13 March2014 Volume78 Number1 MicrobiologyandMolecularBiologyReviews p.89–175 mmbr.asm.org 89 Bräsenetal. PENTOSESYNTHESISPATHWAYSINARCHAEA............................................................................................................127 HexulosephosphateSynthase/PhosphohexuloseIsomerase.............................................................................................129 Ribose-5-PhosphateIsomerase...........................................................................................................................129 Transketolase.............................................................................................................................................132 AlternativePathwaysforC-C Interconversion..........................................................................................................132 5 3 METABOLICANDREGULATORYCHARACTERISTICSOFWELL-STUDIEDARCHAEALMODELORGANISMS..............................................132 RegulationattheTranscriptLevel........................................................................................................................132 RegulationattheProteinLevel...........................................................................................................................133 MetabolicThermoadaptation............................................................................................................................133 Thermococcales(PyrococcusfuriosusandThermococcuskodakarensis)..................................................................................1.34 Growthconditions.....................................................................................................................................134 Sugartransport........................................................................................................................................134 Sugarmetabolism.....................................................................................................................................135 (i)ThemodifiedEMPpathway......................................................................................................................135 D (ii)Gluconeogenesis................................................................................................................................135 o Energetics..............................................................................................................................................137 w Regulationattheproteinlevel.........................................................................................................................137 n Regulationatthegenelevel...........................................................................................................................137 lo TranscriptionalregulatorTrmBinThermococcales.....................................................................................................138 a d TrmB-likeregulatorsinThermococcales................................................................................................................139 e (i)TrmB-likeregulatorsinTco.kodakarensis.........................................................................................................139 d (ii)TrmB-likeregulatorsinPyrococcusfuriosus.......................................................................................................140 f r InvivofunctionofTrmBandTrmB-likeregulatorsinPyrococcus.......................................................................................140 o Sulfolobussolfataricus ....................................................................................................................................140 m Growthconditions.....................................................................................................................................140 h Sugartransport........................................................................................................................................143 t t Sugarmetabolism.....................................................................................................................................143 p : (i)ThepromiscuousbranchedEDpathway.........................................................................................................143 // m (ii)SignificanceofbothEDbranches................................................................................................................144 (iii)Gluconeogenesis................................................................................................................................144 m Energetics..............................................................................................................................................144 b r Regulation.............................................................................................................................................144 . a (i)Regulationattheenzymelevelbyallostericeffectors............................................................................................144 s (ii)Regulationatthetranslationallevel..............................................................................................................145 m (iii)Regulationattheposttranslationallevelbyproteinphosphorylation...........................................................................145 .o (iv)Regulationatthegene/transcriptlevel.........................................................................................................146 r g Transcriptionalregulatorsofthecentralcarbohydratemetabolism...................................................................................146 / Thermoplasmatales(ThermoplasmaacidophilumandPicrophilustorridus) ..............................................................................1.47 o n Growthconditions.....................................................................................................................................147 Sugar(glucose)metabolism...........................................................................................................................147 D Energetics..............................................................................................................................................147 e c Firstinsightsintoregulationattheproteinandgenelevels...........................................................................................147 e Thermoproteustenax .....................................................................................................................................148 m Growthconditions.....................................................................................................................................148 b Sugartransport........................................................................................................................................148 e r Sugar(glucose)metabolism...........................................................................................................................148 1 (i)ThemodifiedEMPpathway......................................................................................................................148 8 (ii)ThebranchedEDpathway.......................................................................................................................150 , 2 (iii)Gluconeogenesis................................................................................................................................150 0 Energetics..............................................................................................................................................150 1 Regulationattheproteinandgenelevels.............................................................................................................150 8 (i)Regulationattheenzymelevelbyallostericeffectors............................................................................................150 b y (ii)Regulationatthegene/transcriptlevel..........................................................................................................151 g Haloarchaea(Haloferaxvolcanii,Haloarculamarismortui,andHalobacteriumsalinarum) ...............................................................1.52 u Growthconditions.....................................................................................................................................152 e Sugartransport........................................................................................................................................152 s t Sugarmetabolism.....................................................................................................................................152 (i)Differentdegradationpathwaysforglucoseandfructose.......................................................................................152 (ii)Glucose-fructosediauxie.........................................................................................................................154 Energetics..............................................................................................................................................154 Regulationatthegeneandproteinlevels.............................................................................................................154 Glycerol-mediatedcataboliterepression..............................................................................................................155 TranscriptionalregulatorGlpR........................................................................................................................1.55 Halobacteriumsalinarum...............................................................................................................................155 (i)TrmB-liketranscriptionalregulator...............................................................................................................155 Methanogens............................................................................................................................................156 (continued) 90 mmbr.asm.org MicrobiologyandMolecularBiologyReviews UniqueFeaturesofArchaealCarbohydrateMetabolism Growthconditions.....................................................................................................................................156 Sugar(endogenousglycogen)metabolism...........................................................................................................157 Energetics..............................................................................................................................................157 Regulationatthegeneandproteinlevels.............................................................................................................157 CONCLUSION...............................................................................................................................................159 ACKNOWLEDGMENTS......................................................................................................................................159 REFERENCES................................................................................................................................................159 SUMMARY archaeal membrane lipids composed of isoprenoid chains ether ThemetabolismofArchaea,thethirddomainoflife,resemblesin linked to sn-glycerol 1-phosphate head groups (i.e., dibiphyta- itscomplexitythoseofBacteriaandlowerEukarya.However,this nyltetraethers or biphytanylethers) rather than fatty acids ester D metaboliccomplexityinArchaeaisaccompaniedbytheabsenceof linkedtosn-glycerol3-phosphate,asfoundinBacteriaandEu- o many “classical” pathways, particularly in central carbohydrate karya(13).Inaddition,murein,thetypicalpeptidoglycanofbac- w metabolism.Instead,Archaeaarecharacterizedbythepresenceof terialcellwalls,isabsentinArchaea,and,forexample,pseudo- nlo unique,modifiedvariantsofclassicalpathwayssuchastheEmb- murein (e.g., Methanosphaera and Methanothermus), S-layer a den-Meyerhof-Parnas(EMP)pathwayandtheEntner-Doudoroff proteins (e.g., Thermoproteus spp. and Sulfolobales), or no cell d e (ED) pathway. The pentose phosphate pathway is only partly envelopes(e.g.,Thermoplasmatales)arefound. d present(ifatall),andpentosedegradationalsosignificantlydiffers Withrespecttotheirlife-styleandmetaboliccomplexity,Ar- fr o fromthatknownforbacterialmodelorganisms.Thesemodifica- chaea resemble Bacteria and lower Eukarya, and chemolithoau- m tions are accompanied by the invention of “new,” unusual en- totrophic, chemoorgangoheterotrophic, as well as phototrophic h zymeswhichcausefundamentalconsequencesfortheunderlying (aerobic/anaerobic)growthhasbeenreported(14,15).However, tt p regulatoryprinciples,andclassicalallostericregulationsiteswell thismetaboliccomplexityinArchaeaisaccompaniedbytheab- : / establishedinBacteriaandEukaryaarelost.Theaimofthisreview senceofmany“classical”pathwaysknownforBacteriaandEu- /m is to present the current understanding of central carbohydrate karya. Instead, Archaea are characterized by the presence of m metabolicpathwaysandtheirregulationinArchaea.Inorderto unique pathways, e.g., methanogenesis, and/or by some unusual, b r giveanoverviewoftheircomplexity,pathwaymodificationsare modified-pathwayversionsoftheclassicalroutes.Thisespeciallyap- .a discussed with respect to unusual archaeal biocatalysts, their plies to central carbohydrate metabolism (CCM). Only modified s m structural and mechanistic characteristics, and their regulatory variantsofclassicalsugardegradationpathwayssuchastheEmbden- . o propertiesincomparisontotheirclassiccounterpartsfromBac- Meyerhof-Parnas(EMP)pathwayandtheEntner-Doudoroff(ED) r g teriaandEukarya.Furthermore,anoverviewfocusingonhexose pathwayhavebeenidentifiedinArchaea.Thepentosephosphate / o metabolic,i.e.,glycolyticaswellasgluconeogenic,pathwaysiden- pathway(PPP)ispresentonlypartly(ifatall),andpentosedeg- n tifiedinarchaealmodelorganismsisgiven.Theirenergygainis radationsignificantlydiffersfromthatknownforbacterialmodel D discussed,andnewinsightsintodifferentlevelsofregulationthat organismssuchasEscherichiacoli.Particularly,themodifications e c have been observed so far, including the transcript and protein anddistinctfeaturesofthesugardegradationpathwayscausefun- e levels(e.g.,generegulation,knowntranscriptionregulators,and damentalconsequencesfortheunderlyingregulatoryprinciples. m b posttranslationalmodificationviareversibleproteinphosphory- Notably,thevastmajorityofmetabolicconversionsand,thus, e lation),arepresented. intermediates found in the glycolytic and also gluconeogenic r 1 pathwaysforglucose-to-pyruvateinterconversionareconserved 8 INTRODUCTION inallthreedomainsoflife,highlightinggovernmentbythermo- , 2 Archaea were established as the third domain of life besides dynamic and chemical constraints (e.g., flux rates/short path- 0 1 BacteriaandEukaryaonly30yearsago(1,2).Todate,most ways)(16).However,inArchaea,theenzymesinvolvedintheir 8 cultivable species are adapted to extreme environments, where conversionoftensharenosimilaritywiththeirbacterialandeu- b y theyconstitutethedominantmajority,orharboruniquemetabolic karyoticcounterpartsandrepresentmembersofnewenzymefami- g capabilitieslikemethanogens.Theso-called“extremophiles”thrive lies. Accordingly, by using a comparative genomics approach, the u e inhostilehabitatscharacterizedbyextremesoftemperature,pH,salt, “central core and variable shell” of archaeal genomes were estab- s or combinations thereof. However, metagenomic/environmental lished,andseveralmissinglinksinarchaealcentralmetabolismwere t molecularbiologyapproachesrevealedthatArchaeaareubiquitous identified,whichareconservedinbacterialandeukaryoticmetabo- andwidelydistributedinmoderatehabitatsandplayamajorrole lism(17,18).Theseidentified“gaps”inarchaealcentralmetabolic ingeochemicalcycles(3–5). pathwaysaretheproductofnonhomologousgenereplacement.In Archaeaexhibitamosaiccharacter,astheysharesometypical themeantime,manyofthesepathwayholeshavebeenclosedbythe bacterial and eukaryotic properties. Their unicellular life-style, identification of the respective candidates by using comparative lack of organelles, cell size and shape, as well as DNA structure genomics-basedapproachesand/orclassicalbiochemistry. (e.g.,onecircularchromosome,operonstructures,andplasmids) Today,267archaealgenomesequenceshavebeencompleted resemblethoseofBacteria,whereasmostmechanismsinvolvedin (asofDecember2013)(GenomeOnlineDatabase[GOLD][http: information processing (e.g., replication, transcription, repair, //genomesonline.org/]), and six main archaeal phyla have been andtranslation)aregenerallyregardedaslesscomplexversionsof proposed: Crenarchaeota, Euryarchaeota, Nanoarchaeota, Thau- the respective eukaryotic equivalents (6–12). However, Archaea marchaeota, Korarchaeota, and the recently proposed Aigar- also possess unique archaeal features, most notably the unique chaeota. March2014 Volume78 Number1 mmbr.asm.org 91 Bräsenetal. The two largest and first-established phyla are the Euryar- thephysiologicalsignificanceofgenescouldbestudiedbymutant chaeotaandtheCrenarchaeota(2).TheEuryarchaeotacompriseall construction(forreviewsandliterature,seereferences27and28). methanogens and extreme halophiles and some thermoacido- Furthermore, transcriptomic (microarray analyses and deep se- philesand(hyper)thermophiles,whereastheCrenarchaeotacon- quencing in combination with reverse transcription-quantitative tain only (hyper)thermophilic species (2). The Nanoarchaeota PCR[qRT-PCR]),proteomic,andmetabolomicaswellassystems compriseonlytwospeciessofar:Nanoarchaeumequitans,which biologyapproacheswerealsousedforArchaea,facilitatingthein- canbegrownonlyincoculturewiththecrenarchaeoteIgnicoccus vestigationofpathwayregulationandunderlyingprinciples. hospitalis,andNst1,wherethelikelyhostisamemberoftheSul- Theaimofthisreviewistopresentthecurrentunderstanding folobales(Acd1)(19,20).TheKorarchaeotainsteadcontainonly ofsugardegradationpathwaysandtheirregulationinArchaea.In uncultivatedhyperthermophilic,anaerobicArchaea,andthehet- ordertogiveanoverviewofthecomplexity,thisreviewissubdi- erotrophic organism “Candidatus Korarchaeum cryptofilum” is videdintotwoparts.Thefirstpartfocusesonpathwaymodifica- thefirstproposedmemberofthisgroup(21,22).Membersofthe tionwithrespecttounusualarchaealbiocatalysts,theirstructural Thaumarchaeota are both thermophiles and mesophiles and andmechanisticcharacteristics,andtheirregulatorypropertiesin D thrive in very different environmental niches (e.g., freshwater, comparison to their classic counterparts from Bacteria and Eu- o w soil,ocean,andhotsprings)(23,24).Allmembersofthisphylum karya.Inthesecondpart,anoverviewfocusingonhexosemeta- n characterizedsofar(e.g.,Nitrososphaeragargensis,Nitrosopumilus bolicpathways,i.e.,glycolysisaswellasgluconeogenesis,identi- lo maritimus, and Cenarchaeum symbiosum) are chemolithoau- fied in archaeal model organisms is given. Their energy gain is a d totrophicammoniaoxidizers,whichwasthoughttobeperformed discussed,andnewinsightsintothedifferentlevelsofregulation e d solely by Betaproteobacteria and Gammaproteobacteria (24). thathavebeenobservedsofar,includingthetranscriptandpro- f HWCG1istheonlyidentifiedmemberoftheAigarchaeotasofar teinlevels(e.g.,generegulation,knowntranscriptionregulators, ro (25). However, whether the Aigarchaeota are indeed a new ar- andposttranslationalmodification[PTM]viareversibleprotein m chaealphylumoradeeplybranchingthaumarchaeallineageisstill phosphorylation),arepresented(forpreviousreviewsinthefield h t amatterofdebate(26). of archaeal central carbohydrate metabolism, see references 14 tp WhereasthecarbohydratemetabolismofmembersoftheCre- and29–37)(seeFig.1and12forthemostfrequentlyusedabbre- :/ / narchaeota and Euryarchaeota has been studied in considerable viationsforenzymesandmetabolicintermediates). m m detail, only little information is available for the remaining ar- chaealphyla.InArchaea,sugardegradationwasfirstinvestigated MODIFICATIONSOFTHEEMBDEN-MEYERHOF-PARNAS br inaerobicextremehalophiles,suchasHalobacteriumsp.andHa- PATHWAYINARCHAEA .a s loarcula, and thermophilic acidophiles, e.g., the euryarchaeon TheEMPpathwaycanberegardedasanevolutionarilyoptimized m ThermoplasmaacidophilumandthecrenarchaeonSulfolobussol- pathwayfortheconversion/oxidationofglucoseto2moleculesof .o fataricus. In the early 1990s, anaerobic hyperthermophiles, in- pyruvate,yieldingATPaswellasreducingequivalentsandinter- rg cludingthefermentativelygrowingeuryarchaeonPyrococcusfu- mediatesasprecursorsforcellularbuildingblocks.Pathwayopti- / o riosus and the crenarchaeon Thermoproteus tenax, growing on mizationmeansthatproductformationisachievedinthefewest n sugarsbymeansofsulfurrespiration,werealsoextensivelyinves- possiblestepswithoverallexergonismtodrivethewholeprocess. D e tigated,andthesestudieshavebeenextendedtootheranaerobic Theaimistoensurethehighestpossibleeffectivity/fluxesonthe c hyperthermophilessuchasmembersoftheEuryarchaeota(Ther- onehandandtoenablethehighestpossibleenergy/ATPyieldson e m mococcusspp.andArchaeoglobusfulgidus)andtheCrenarchaeota theotherhand.Thesepurposesareachievedundertheconsider- b (Desulfurococcus amylolyticus and the [micro]aerophilic/aerobic ationofchemicalandmechanisticfeasibilitiesaswellasthephys- e r organismsPyrobaculumaerophilumandAeropyrumpernix).Also, icochemicalpropertiesofintermediateswithrespectto,e.g.,sta- 1 the most acidophilic organism known to date, the moderately bility,permeability,polarity,andtoxicity(16,38). 8 , thermophiliceuryarchaeonPicrophilustorridus,hasbeeninvesti- In the classical EMP pathway, glucose is phosphorylated to 2 0 gated,andtheroleofsugar-metabolizingroutesinsomemetha- glucose6-phosphate(G6P)eitherbyATP-dependenthexokinase 1 nogenic organisms, such as Methanococcus maripaludis and (HK)/glucokinase(GLK)or,inmanyBacteria,bytheactionofthe 8 Methanocaldococcusjannaschii,havebeenaddressed. phosphoenolpyruvate (PEP) phosphotransferase system (PTS). b y Byusing13Cnuclearmagneticresonance(NMR)analyses,the G6Pisthenisomerizedtofructose6-phosphate(F6P)viaphos- g questionofpathwayutilization,i.e.,whethertheEMPpathway, phoglucoseisomeraseandisagainphosphorylatedtoformfruc- u e theEDpathway,orbothroutesareutilizedforsugardegradation tose 1,6-bisphosphate (F1,6BP), a key intermediate of the EMP s t in the respective organisms, and to which extent, has been ad- pathway, by phosphofructokinase. F1,6BP is cleaved to dihy- dressed.Withclassicalbiochemicaltechniqueslikeenzymemea- droxyacetone phosphate (DHAP) and glyceraldehyde 3-phos- surementsincrudeextracts,nativeproteinpurification,aswellas phate(GAP),catalyzedbyF1,6BPaldolase.DHAPisconvertedto comparative genome analyses and with molecular biological GAPviatriosephosphateisomerase(TIM),andGAPisoxidizedto methodslikerecombinantexpressionofcandidateenzymes,the 1,3-bisphosphoglycerate(1,3BPG)throughtheactionofglyceral- pathways could be reconstructed. The enzymes involved in the dehyde-3-phosphate dehydrogenase (GAPDH), yielding NA- pathwayshavebeenidentifiedandcharacterized,andthestruc- D(P)H.Thephosphatemoietyfromtheacidanhydride1,3BPGis turesofmoreandmoreofthearchaealenzymesinvolvedinsugar transferredtoADP,yieldingATPinthefirststepofsubstrate-level metabolismhavebeendetermined.Also,“genetictoolboxes”fora phosphorylationwithinthepathway,catalyzedbyphosphoglyc- number of archaeal model organisms, e.g., Haloferax volcanii, erate kinase (PGK). The resulting 3-phosphoglycerate (3PG) is Thermococcus kodakarensis, Sul. solfataricus, Sulfolobus acidocal- further converted to 2-phosphoglycerate (2PG) and the phos- darius,andPyr.furiosus,aswellasmethanogenssuchasMetha- phoenolesterPEPviaphosphoglyceratemutase(PGAM)andeno- nococcusspp.andMethanosarcinaspp.havebeenestablished,and lase(ENO).FromPEP,thephosphatemoietyisagaintransferred 92 mmbr.asm.org MicrobiologyandMolecularBiologyReviews UniqueFeaturesofArchaealCarbohydrateMetabolism D o w n lo a d e d f r o m h t t p : / / m m b r . a s m . o r g / o n D e c FIG1GlucosedegradationviatheEMPpathwayknownformostBacteriaandEukarya(classical)andthemodifiedEMPversionsreportedforArchaea. e Allostericallyregulatedenzymesaredepictedinred.Abbreviations:ENO,enolase;FBPA,fructose-1,6-bisphosphatealdolase;GAPDH,glyceraldehyde-3- m phosphatedehydrogenase;GAPN,nonphosphorylatingGAPDH;GAPOR,GAP:Fdoxidoreductase;GLK,glucosekinase;HK,hexokinase;PEPS,PEPsynthe- b e tase;PFK,phosphofructokinase;PGI,phosphoglucoseisomerase;PGI/PMI,phosphoglucoseisomerase/phosphomannoseisomerase;cPGI,cupin-typephos- r phoglucose isomerase; PGAM, phosphoglycerate mutase (dPGAM, 2,3-bisphosphoglycerate [2,3BPG] cofactor dependent; iPGAM, 2,3BPG cofactor 1 independent);PK,pyruvatekinase;PPDK,pyruvate:phosphatedikinase;ROK,hexokinaseoftherepressorprotein,openreadingframe,sugarkinasefamily; 8 , G6P,glucose6-phosphate;F6P,fructose6-phosphate;F1,6BP,fructose1,6-bisphosphate;DHAP,dihydroxyacetonephosphate;GAP,glyceraldehyde3-phos- 2 phate;1,3BPG,1,3-bisphosphoglycerate;3PG,3-phosphoglycerate;2PG,2-phosphoglycerate;PEP,phosphoenolpyruvate. 0 1 8 toADPviasubstrate-levelphosphorylationtofinallyformpyru- Duetoitscomparativelyhighenergyyieldofmaximally2mol b y vateinthepyruvatekinase(PK)-catalyzedreaction(foranover- ATP/molofglucose,theutilizationoftheEMPpathwayispartic- g view,seeFig.1).Thus,theconversionof2molGAPtopyruvate ularlyadvantageousfororganismswithananaerobicorfaculta- u e yields4molATPinthePGKandPKreactions,and2molATPhas tivelyanaerobiclife-stylegainingenergymainlybyfermentative s tobeinvestedforphosphorylationofglucoseandF6Pinthepre- metabolismfromsugars/glucose.Accordingly,byusing13CNMR t paratoryphaseofglycolysis,resultinginanetyieldof2molATP studiesincombinationwithenzymemeasurementsincrudeex- permolglucoseoxidizedtopyruvate.Withinthispathway,phos- tracts, the anaerobic hyperthermophiles, the Euryarchaeota Pyr. phorylationfromglucosetoG6PandfromF6PtoF1,6BPaswell furiosusandThermococcusspp.,andthecrenarchaeonDesulfuro- aspyruvateformationfromPEPrepresentirreversiblesteps,and coccusfermentativelygrowingonsugarshavebeenshowntode- theenzymesinvolvedareaccordinglythemainpointsofallosteric gradeglucose100%viamodifiedEMPpathways.Also,thecren- regulation.Also,theseirreversiblereactions,especiallythePKand archaeonTpt.tenax,growingonglucoseusingsulfurreduction, phosphofructokinase(PFK)reactions,havetobebypassedduring metabolizesthesugar85%viaanEMPpathwaymodification(39, gluconeogenesis.ThisisusuallyaccomplishedbyPEPsynthetase 40).Fortheanaerobic,euryarchaealsulfatereducerArc.fulgidus (PEPS), pyruvate:phosphate dikinase (PPDK), or the pyruvate aswellasthemicroaerophilicCrenarchaeotaPyb.aerophilumand carboxylase/PEPcarboxykinase(PCK)enzymecoupleforthePK Aer.pernix,modifiedEMPversionshavealsobeendescribedon reactionandfructosebisphosphatase(FBPase)forthePFKreac- thebasisofenzymeanalyses(41,42).Underliningtheoptimized tion. propertiesoftheEMPpathwayandthethermodynamic,kinetic, March2014 Volume78 Number1 mmbr.asm.org 93 Bräsenetal. and mechanistic constraints under which the pathway evolved, archaealEMPpathwayversionsaswellastheclassicalEMPpath- glucosedegradationviathesemodifiedEMPpathwayversionsin wayfromBacteriaandEukarya). Archaeabasicallyproceedsviathesameintermediatesknownfor theclassicalEMPpathwayinBacteriaandEukarya,alsohighlight- GlucosePhosphorylation ing the ancestral character of the pathway. However, although ThefirststepinthemodifiedEMpathwaysofArchaeaisthephos- catalyzing the same conversions, the enzymes in archaeal EMP phorylation of glucose to glucose 6-phosphate. In Bacteria, this pathwayversionsdifferremarkably(seeFig.1foracomparative process is coupled to transport via the PEP phosphotransferase illustration of the classical EMP pathway and the modified ar- systemoriscatalyzedbyglucoseandATP-specificglucokinases, chaealversions).Thepathwaymodificationsoccurmainlyinthe whereas in Eukarya, this reaction is catalyzed by ATP-specific upper part of the archaeal pathways, involving ADP-, PP-, or hexokinases,whichusuallyshowbroadsubstratespecificityfora i ATP-dependent kinases, distinct from the classical enzymes in varietyofothersugars,likefructose,mannose,andgalactose(44, BacteriaandEukarya.Also,thephosphoglucoseisomerasesaswell 45).Conversely,thebacterialATP-dependentglucokinasesshow asthefructose-1,6-bisphosphatealdolasesinArchaeadifferfrom apronouncedspecificityfortheirrespectivesugarsubstrates(for D theirbacterialandeukaryoticcounterparts.Themoststrikingdif- literature,seereference 46).Boththeeukaryotichexokinasesas o w ferenceinarchaealpathways,especiallyin(hyper)thermophiles,is well as the bacterial glucokinases constitute distinct families n the direct and irreversible oxidation of GAP to 3PG, catalyzed within the actin-like ATPase domain superfamily. Members of lo either by NAD(P)(cid:3)-dependent nonphosphorylating glyceralde- thissuperfamilyshareasimilaroverallfoldconsistingofalarge a d hyde-3-phosphate dehydrogenase (GAPN) or, mainly in anaer- domain(five-strandedmixed(cid:4)sheetfacedbyfive(cid:2)helicesonone e obes,byferredoxin(Fd)-dependentglyceraldehyde-3-phosphate sideofthesheet)andasmalldomain(five-strandedmixed(cid:4)sheet d oxidoreductase(GAPOR).Bothenzymesomittheformationof surroundedbythree(cid:2)helices,twoononesideandoneonthe fro 1,3-bisphosphoglycerateandtheproductionofATPviasubstrate- othersideofthesheet).Theactivesiteislocatedatthedomain m levelphosphorylation,withconsiderableconsequencesforpath- interface,anduponsubstratebinding,theconformationchanges h t wayenergetics.Reducedferredoxin(Fdred)canserveasanelec- toitsclosedstatethroughdomainmovement(44).Onthebasisof tp tron donor in anabolic reactions instead of or at least in crystalstructures(47–49)andtheresultsofmutagenesisstudies :/ / combination with NAD(P)H. The utilization of ferredoxin also (50),thecatalyticmechanismforsugarkinasesfromtheactin-like m m allows for additional energy conservation via H generation by ATPasedomainsuperfamilyhasbeenproposedtoinvolveacon- 2 b meansofmembrane-boundhydrogenase,whichisespeciallyad- servedAsp(i.e.,Asp657inhumanhexokinaseI)thatabstractsthe r . vantageous under anaerobic conditions. Despite the energetics, protonfromthe6-hydroxylgroupofglucoseasacatalyticbase. a s theEMPpathwaymodificationsinArchaeaalsostronglyinfluence Thisenablesanucleophilicattackofthe(cid:5)-phosphorusofATPon m theirregulation.TheclassicalsitesofallostericregulationinBac- theactivated6-oxygenofglucose,finallyyieldingG6P(seeFig.3). .o teria and Eukarya, i.e., particularly HK and ATP-PFK (PFK-A) ThepositivechargesofArg539andaMg2(cid:3)ionareproposedto rg (seebelow),arereplacedinarchaealpathways.Also,thearchaeal stabilize the reaction intermediate. These catalytic residues are / o PKsshowstronglyaltered(ifany)regulatoryproperties(seebe- structurally conserved in the sugar kinases of the actin-like n low).Instead,asknownsofar,GAPN,mainlyfoundin(hyper)- ATPase domain superfamily. In Archaea, two different mecha- D e thermophilicArchaea,representsthemainsiteofallostericregu- nismsofglucosephosphorylationhavebeendescribed. c lation. ADP-dependentglucokinase.InEuryarchaeotasuchasPyr.fu- e m Asoutlinedbelow,theunusualenzymesinthearchaealpath- riosus,Thermococcuslitoralis,andArc.fulgidus,glucosephosphor- b waymodificationsrepresentmostlyexamplesofnonhomologous ylation is carried out by ADP-dependent glucokinases (ADP- e r genereplacementsratherthannovelinventions(30).Incontrast GLKs).Theseenzymesshowhighspecificityforglucose(51–55) 1 tothemodificationsintheupperpartofthearchaealEMPvari- andbelongtotheribokinasesuperfamily.Conversely,intheCre- 8 , ants,theenzymesinthelowerpart,withtheexceptionofcatabolic narchaeotaTpt.tenax,Pyrobaculumislandicum,Pyb.aerophilum, 2 0 GAPORandGAPN,i.e.,TIM,PGAM,ENO,andPK,arehomol- Aer.pernix,andDes.amylolyticus,thisprocessisATPdependent 1 ogoustotheirclassicalcounterpartsinBacteriaandEukarya.Also, (ADPforming),involvinganATP-dependenthexokinasewitha 8 theGAPDH/PGKcouple,whichispresentinArchaea(exceptfor broad hexose substrate spectrum, as shown for the enzymes of b y extremehalophilesandsomemethanogens)exclusivelyforana- Aer.pernixandTpt.tenax(40,41,46,55).TheseATP-dependent g bolicpurposes(seeGluconeogenesis,below),correspondswellto archaealhexokinasesbelongtotheROK(repressorprotein,open u e itsclassicalcounterparts.Furthermore,thephylogeniesofthese readingframe,sugarkinase)family(seebelow).Whereasclassical s t universally distributed enzymes show distinct archaeal clusters, eukaryotichexokinasesrepresentmonomeric,andsometimesdi- whereasthebacterialandeukaryoticproteinsappearedtobemore meric,proteins(subunitsizeof(cid:6)50or100kDa),andthebacterial closelyrelatedandlessdistinct,presumablyduetoanendosym- ATP-dependentglucokinasesaredimerscomposedof(cid:6)30-kDa biotic origin and lateral gene transfer events, respectively. The subunits,thearchaealADP-GLhavebeenbiochemicallycharac- universaldistributionandhomologyoftheenzymesofthelower terizedasmonomericenzymes(subunitof(cid:6)50kDa)(44–46,51, partoftheglycolysispathwayincombinationwiththeirlowevo- 53–56). lutionary rates have been taken as a strong indication that the ArchaealADP-GLKspreferADPasthephosphoryldonorand EMPpathwayevolvedinthegluconeogenicdirection(43). additionallyalsoutilizeCDPbutnotGDP,IDP,orUDP(57).The Inthefollowingsections,wedescribestepbystepthearchaeal Arc.fulgidusenzymeappearstobespecificforADP(51,53,54). biocatalystsoftheupperandthelowershuntsoftheEMPpathway The bifunctional ADP-GLK/PFK from Mca. jannaschii also uti- incomparisontotheirbacterialandeukaryoticcounterparts,with lizesGDP(40%)inadditiontoADP(100%)and,lessefficiently, specialreferencetostructuralandmechanisticcharacteristics(Ta- CDP (14%) (58). The crystal structures of archaeal ADP-GLKs ble1givesanoverviewofalltheenzymesinvolvedinthemodified from Pyr. furiosus, Pyrococcus horikoshii, and Tco. litoralis have 94 mmbr.asm.org MicrobiologyandMolecularBiologyReviews UniqueFeaturesofArchaealCarbohydrateMetabolism beensolved,identifyingthearchaealADP-GLKsasmembersof andBac.subtilis)orhomotetramers(The.thermophilusandStr. theADP-dependentsugarkinasefamilywithintheribokinasesu- griseus).SincemostbacterialROKkinaseswereshowntobespe- perfamily (59–61). The basic fold of ADP-GLK is composed of cificfortheirrespectivesugarsubstrates,ithasbeendiscussedthat onelargedomainandanadditionalsmalldomain,withtheactive thebroadsubstratespectrummightbeacharacteristicfeatureof site located at the domain interface (Fig. 2A). The nucleotide archaealROKsugarkinases(63).However,theThe.thermophilus bindingsitelieswithinthelargedomain,andthesugarbinding ROKsugarkinasealsoconvertsmannoseinadditiontoglucose siteliesinacleftbetweenbothdomains.Thelargerdomaincon- (64).AlthoughnocrystalstructuresofarchaealROKkinasesare sistsofatwisted11-to12-stranded(cid:4)sheetflankedonbothfaces available,thestructuresfromBacteriarevealedatwo-domainar- by13(cid:2)helicesand33 helices,formingan(cid:2)/(cid:4)3-layersandwich. chitectureandfoldsimilartothosedescribedforhexokinasesand 10 Thesmallerdomain,whichcoverstheactivesite,formsan(cid:2)/(cid:4) ATP-dependent glycerate kinases (ATP-GKs), with both sub- two-layerstructurecontaining5to7(cid:4)strandsand4(cid:2)heliceson stratesbindinginthecleftbetweenbothdomains(64,66).From thefarsideoftheactive-sitecleftofthesheet(56,59–61).Thelarge theaminoacidsequencesimilarities,comparablestructuresmight domainrepresentstheribokinasecorefold,whichtypicallycon- alsobeexpectedforthearchaealROKkinases.ThearchaealROK D sistsofaneight-stranded(cid:4)sheetsurroundedbyeight(cid:2)helices, hexokinaseactivitiesappearedtobedependentonbivalentmetal o w threeononesideandfiveontheothersideofthesheet(57).Upon ions (46, 55), and in the crystal structures of bacterial ROK ki- n sugar binding, the small domain undergoes a conformational nases,aboundZn2(cid:3)ioninazincfingermotifhasbeenobserved lo change, closing the active site (59, 61). Although the “classical” (64, 66). This zinc ion interacts with residues that in turn are a d hexokinasesinEukaryahavebeendescribedtopossessasimilar involvedinsubstratebinding,andthereby,thiszincionhelpsto e d two-domain architecture and to follow a comparable catalytic positionthesugarsubstrateforcatalysis.Thecatalyticmechanism f mechanism, there are no similarities between ADP-GLKs and oftheROKkinasesseemstobesimilartothatdescribedforATP- ro hexokinases either on the sequence level or in the fold of both GLKandHK(65).Althoughasimilarmechanismwasalsopro- m domains (44, 45, 57). ADP-GLKs contain two sequence motifs, posedforADP-dependentkinases(seeabove),therearenostruc- h t i.e., GXGD and NXXE, highly conserved in members of the ri- turalorsequencesimilaritiesbetweenbothenzymes,andalso,the tp bokinasesuperfamilyandinvolvedincatalysis(57).Theaspartate mechanismofmetaliondependencyappearstobedifferent(46, :/ / residue of the GXGD motif has been proposed to act in sugar 50) (Fig. 2A and C and 3). In the crystal structure of the Bac. m m bindingandasageneralbaseduringthecatalyticcycle(proton subtilisROKfructokinase,nodrasticconformationalchanges b abstractionfromtheacceptorhydroxylgroup)(57,59)(Fig.3). havebeenobserveduponsubstratebinding. r . AsparagineandglutamateoftheNXXEmotifareproposedtobe Otherarchaealsugarkinases.ForSulfolobustokodaii,ahexoki- a s involvedinbivalentmetalcationandnucleotidebinding(57,62). nase from the actin-like ATPase domain superfamily has been m ADP-GLKsdependstrictlyonbivalentmetalions,whichcoordi- purified,andtheencodinggenehasbeenidentified(67).The64- .o natepropernucleotidebindingandpositioningofthephosphate kDadimericenzyme(subunitsof32kDa)showedabroadsub- rg groupsforthephosphoryltransferreaction(51,53,54,57,62). stratespectrum,convertingmannose,glucosamine,N-acetylglu- / o Recently,athirdmotif(HXE),withahighlyconservedglutamate cosamine(GlcNAc),and2-deoxyglucoseinadditiontoglucose, n involvedinmetalioncoordination,hasbeenidentified.Further- withapreferenceforATPasthephosphoryldonor.ADPshowed D e more, binding of a second metal ion to another conserved se- aninhibitoryeffectontheenzyme.ThecrystalstructureofSul. c quence motif in ADP-dependent sugar kinases with regulatory tokodaiiHK(StHK)showedahexokinase-likefold(Fig.2C)and e m implicationshasbeenproposed(62).However,alloftheADP- exhibited some sequence similarities with mammalian GlcNAc b dependentsugarkinases,includingthosefromArchaeareported kinases,althoughthelatterenzymesarespecificforGlcNAc(49). e r sofar,didnotshowanyregulatorypropertieslikecooperativityor HomologsofthisSul.tokodaiihexokinasewithsequencesimilar- 1 allosteric control, thus differing from eukaryotic hexokinases, itiesof(cid:7)50%wereidentifiedonlyinSulfolobusspecies.Dueto 8 , whichrepresentoneofthesitesofallostericcontrolintheclassical theirfoldandsincetheseSulfolobusenzymesseemtobelongto 2 0 EMPpathway(56). neithertheROKnortheglucokinasefamily,ithasbeendiscussed 1 ROK hexokinase. The second mechanism of glucose phos- thattheyrepresentnewmembersofthehexokinasefamilywitha 8 phorylation in Archaea is catalyzed by ATP-dependent kinases uniquesubstratespecificity. b y withbroadsugarsubstratespecificity,asdescribedforAer.pernix InhalophilicArchaea,amodifiedversionoftheEMPpathway g and Tpt. tenax, which convert several other sugars in addition isusedforthedegradationoffructose,inwhichthesubstrateis u e to glucose, such as fructose, mannose, and 2-deoxyglucose phosphorylatedtofructose1-phosphate(F1P)viaketohexokinase s (46, 55). These (cid:6)35-kDa monomeric enzymes exhibit the (KHK).F1Pisfurtherphosphorylatedby1-phosphofructokinase t two signature patterns of the ROK family [LIVM]-x -G- (1-PFK)toF1,6BP,whichisthencleavedtoDHAPandGAPby (2) [LIVMFCT]-G-X-[GA]-X-G-X -[GATP]-X -G-[RKH] and fructose bisphosphate (FBP) aldolase (FBPA). DHAP and GAP (3-5) (2) C-X-C-GX -G-X-[WILV]-E-X-[YFVIN]-X-[STAG] (46). The areconvertedtopyruvatebyclassicalEMPpathwayenzymes(68– (2) ROKkinasesconstituteonefamilywithinthesuperfamilyofac- 70).TheKHKfromHaloarculavallismortiscatalyzingthefirststep tin-likeATPasedomainproteinsalsocomprisingthehexokinase inthisEMPpathwaymodification,i.e.,theATP-dependentphos- family and the ATP-dependent glucokinase family (see above) phorylationoffructosetofructose1-phosphate,hasbeenpurified (44).InadditiontothearchaealROKhexokinases,severalROK andcharacterized(71,72).Thenativemolecularmasswasdeter- sugarkinaseshavebeencharacterized(e.g.,fromThermotogama- minedtobe100kDa,butthesubunitcompositionandsizescould ritima),andcrystalstructures(e.g.,fromThermusthermophilus, notunambiguouslybeestablished.F1P-formingKHKsareknown Bacillussubtilis,andStreptomycesgriseus)havebeenreported(63– mainlyfromEukarya.TheseenzymesbelongtothePFK-Bfamily, 65).IncontrasttothemonomericarchaealROKkinases,thebac- as revealed by sequence and structural analyses, and represent terialROKsugarkinasesrepresenthomodimers(Tmt.maritima (cid:6)70-kDadimericproteinscomposedofasingle(cid:6)35-kDasub- March2014 Volume78 Number1 mmbr.asm.org 95 Bräsenetal. ea Distribution EukaryaBacteria Archaea(mainlyanaerobicEuryarchaeota) Archaea(Crenarchaeota),BacteriaHalophilicEuryarchaeota,Eukarya Crenarchaeota(Sulfolobus) Eukarya,Bacteria,halophilicEuryarchaeota,methanogenicEuryarchaeota(Methanococcus)Crenarchaeota,Euryarchaeota(Thermoplasma) AnaerobicEuryarchaeota(includingMethanosarcinasomeBacteria(Ensifermeliloti,mazei),SalmonellaTyphimurium,horizontalgenetransfer)Eukarya,Bacteria Archaea,(mainlyanaerobicEuryarchaeota),glycogen-formingmethanogensCrenarchaeota(Aeropyrumpernix,Desulfurococcusamylolyticus)Crenarchaeota(Thermoproteustenax),someBacteria,Eukarya(protists,plants) Eukarya Archaea,Bacteria HalophilicEuryarchaeota,Bacteria,fungi Eukarya,Bacteria,Archaea Eukarya,Bacteria,halophilicEuryarchaeota,glycogen-degradingmethanogens(catabolicandanabolic),allotherArchaea(onlyanabolic) Eukarya,Bacteria,halophilicEuryarchaeota,glycogen-degradingmethanogens(catabolicandanabolic),allotherArchaea(onlyanabolic) D a athwayversionsinArch Proteinfamily(ies) HexokinasefamilyGlucokinasefamily ADP-dependentsugarkinasefamilyROKfamilyPFK-B ProbablyBadF/BadG/BcrA/BcrDATPasefamilyPGIfamily PGI/PMIfamily cPGIfamily Phosphofructokinasefamily(knownasPFK-A)ADP-dependentsugarkinasefamilyPFK-Bfamily Phosphofructokinasefamily(knownasPFK-A),formingadistinctsubfamilyClassIaldolasefamily ClassIaldolasefamily ClassIIFBPaldolasefamily TriosephosphateisomerasefamilyGAPDHN-andC-terminaldomainfamilies,respectively Phosphoglyceratekinasefamily ownloaded from wellasinthemodifiedEMPp Proteinsuperfamily(ies)(accordingtotheSCOPdatabase) Actin-likeATPasesuperfamilyActin-likeATPasesuperfamily Ribokinasesuperfamily Actin-likeATPasesuperfamilyProteinsequenceinArchaeaunknown;eukaryoteribokinasesuperfamilyActin-likeATPasesuperfamily PGIsuperfamily(SISdomainsuperfamily)PGIsuperfamily(SISdomainsuperfamily)Cupinsuperfamily Phosphofructokinasesuperfamily Ribokinasesuperfamily Ribokinasesuperfamily Phosphofructokinasesuperfamily (cid:4)(cid:2)Aldolasesuperfamily[()barrel8fold] (cid:4)(cid:2)Aldolasesuperfamily[()barrel8fold] (cid:4)(cid:2)Aldolasesuperfamily[()barrel8fold] Triosephosphateisomerase(cid:4)(cid:2)superfamily[()barrelfold]8N-terminaldomainNAD(P)bindingRossmanfold-likedomainsuperfamily;C-terminalglyceraldehyde3-phosphatedehydrogenase-like,C-terminaldomainsuperfamilyPhosphoglyceratekinasesuperfamily http://mmbr.asm.o PpathwayinBacteriaandEukaryaas Reaction(cid:3)¡(cid:3)HexoseATPhexose6-phosphateADP(cid:3)¡(cid:3)GlucoseATPglucose6-phosphateADP(cid:3)¡(cid:3)GlucoseADPglucose6-phosphateAMP(cid:3)¡(cid:3)HexoseATPhexose6-phosphateADP(cid:3)¡(cid:3)FructoseATPfructose1-phosphateADP (cid:3)¡(cid:3)HexoseATPhexose6-phosphateADP Glucose6-phosphatefructose6-phosphate% Glucose6-phosphate/mannose6-phosphatefructose6-phosphate%Glucose6-phosphatefructose6-phosphate% (cid:3)¡Fructose6-phosphateATPfructose1,6-(cid:3)bisphosphateADP(cid:3)¡Fructose6-phosphateADPfructose1,6-(cid:3)bisphosphateAMP(cid:3)¡Fructose6-phosphateATPfructose1,6-(cid:3)bisphosphateADP(cid:3)Fructose6-phosphatePPfructose1,6-%i(cid:3)bisphosphatePi Fructose1,6-bisphosphate%(cid:3)dihydroxyacetonephosphateglyceraldehyde3-phosphateFructose1,6-bisphosphate%(cid:3)dihydroxyacetonephosphateglyceraldehyde3-phosphateFructose1,6-bisphosphate%(cid:3)dihydroxyacetonephosphateglyceraldehyde3-phosphateDihydroxyacetonephosphate%glyceraldehyde3-phosphate(cid:3)(cid:3)(cid:3)Glyceraldehyde3-phosphateNAD(P)Pi(cid:3)(cid:3)1,3-bisphosphoglycerateNAD(P)H%(cid:3)H (cid:3)1,3-BisphosphoglycerateADP3-%(cid:3)phosphoglycerateATP on December 18, 20rg/ M n 1 assicalE Abbreviatio HKATP-GLK ADP-GLK ROK-HKKHK HK PGI PGI/PMI cPGI ATP-PFK ADP-PFK PFK-B PP-PFKi FBPAI FBPAIA FBPAII TIM GAPDH PGK 8 by g hecl (cid:3)), ue volvedint Cno. 7.1.17.1.2 7.1.147 7.1.17.1.3 7.1.1 3.1.9 3.1.9/5.3.1.8 3.1.9 7.1.11 7.1.146 7.1.11 7.1.90 1.2.13 1.2.13 1.2.13 3.1.1 2.1.13(NADP1.2.1.12(cid:3)(NAD) 7.3.2 st n E 2.2. 2. 2.2. 2. 5. 5. 5. 2. 2. 2. 2. 4. 4. 4. 5. 1. 2. i s TABLE1Overviewofenzyme Enzyme HexokinaseATP-dependentglucokinase ADP-dependentglucokinase ROKhexokinaseKetohexokinase Hexokinase Phosphoglucoseisomerase Phosphoglucose/phosphomannoseisomeraseCupin-typephosphoglucoseisomerase ATP-dependentphosphofructokinase ADP-dependentphosphofructokinase ATP-dependentphosphofructokinase PP-dependentphosphofructokinasei Fructose-1,6-bisphosphatealdolaseclassI ArchaealtypeclassIfructose-1,6-bisphosphatealdolase Fructose-1,6-bisphosphatealdolaseclassII Triosephosphateisomerase Glyceraldehyde-3-phosphatedehydrogenase(phosphorylating) Phosphoglyceratekinase 96 mmbr.asm.org MicrobiologyandMolecularBiologyReviews UniqueFeaturesofArchaealCarbohydrateMetabolism a n GAPN hanosarci (Hyper)thermophilicArchaea AnaerobicArchaea,someanaerobic(hyper)thermophilesinadditionto Vertebrates,yeasts,Bacteria,mainlythermoacidophilicArchaeaandMetspp.Plants,nematodes,Bacteria,Archaea Eukarya,Bacteria,Archaea Eukarya,Bacteria,Archaea Eukarya,Bacteria,Archaea D o ALDH-likefamily Aldehyde:ferredoxinoxidoreductase,N-andC-terminaldomainfamilyCofactor-dependentphosphoglyceratemutasefamily2,3-Bisphosphoglycerate-independentphosphoglyceratemutase,catalyticdomainfamily;2,3-bisphosphoglycerate-independentphosphoglyceratemutase,substratebindingdomainfamilyEnolase-like,N-andC-terminaldomain-likefamilyPyruvatekinasefamily(N-terminaldomain);pyruvatekinasebetabarreldomainfamily(Cterminal)Pyruvate:phosphatedikinaseN-terminal,central,andC-terminaldomainfamilies,respectively(alsoknownasPEP-utilizingenzymefamily) wnloaded from ALDH-likesuperfamily Aldehyde:ferredoxinoxidoreductase,N-andC-terminaldomainsuperfamily Phosphoglyceratemutase-likesuperfamily Alkalinephosphatasesuperfamily;2,3-bisphosphoglycerate-independentphosphoglyceratemutase,substratebindingdomainsuperfamily Enolase-like,N-andC-terminaldomainsuperfamily PEP-pyruvatedomainsuperfamily;pyruvatekinasebeta-barreldomain-likesuperfamily GlutathionesynthetaseATPbindingdomain-likesuperfamily(N-terminaldomain)(ATPgraspfold);phosphohistidinedomainsuperfamily(centraldomain);phosphoenolpyruvate/pyruvatedomainsuperfamily(C-terminaldomain)(TIMbarrelfold) http://mmbr.asm.o r (cid:3)(cid:3)¡Glyceraldehyde3-phosphateNAD(P)(cid:3)(cid:3)(cid:3)3-phosphoglycerateNAD(P)HH (cid:3)¡Glyceraldehyde3-phosphateFd3-ox(cid:3)aphosphoglycerateFdred 3-Phosphoglycerate2-phosphoglycerate% 3-Phosphoglycerate2-phosphoglycerate% 2-Phosphoglyceratephosphoenolpyruvate% (cid:3)¡(cid:3)PhosphoenolpyruvateADPpyruvateATP (cid:3)(cid:3)PyruvateATPP%i(cid:3)(cid:3)phosphoenolpyruvateAMPPPi on December 18, 20g/ 1 8 GAPN GAPOR dPGAM iPGAM ENO PK PPDK by g u e n. s oxi t d 1.9 7.6 2.1 2.1 1.11 1.40 9.1 ferre 1.2. 1.2. 5.4. 5.4. 4.2. 2.7. 2.7. uced Glyceraldehyde-3-phosphatedehydrogenase(nonphosphorylating)Glyceraldehyde-3-phosphate:ferredoxinoxidoreductase 2,3-Bisphosphoglycerate-dependentphosphoglyceratemutase 2,3-Bisphosphoglycerate-independentphosphoglyceratemutase Enolase Pyruvatekinase Pyruvate:phosphatedikinase aFd,oxidizedferredoxin;Fd,redoxred March2014 Volume78 Number1 mmbr.asm.org 97 Bräsenetal. identifiedand,sincefructosewasshowntobetransportedintothe cellsandphosphorylatedbyaPTS(seebelow)inHfx.volcanii,the activityofKHKmightnotberequired.Nevertheless,inHaloar- cula marismortui, KHK activity seems to be induced during growthonfructose(70). PhosphoglucoseIsomerase Phosphoglucose isomerases (PGIs) catalyze the interconversion of the aldose G6P to the ketose F6P in both the classical EMP pathwayinBacteriaandEukaryaaswellasthemodifiedversions inArchaea.Thisreactionisanintramolecularredoxreaction/elec- tronrearrangementinwhichanaldehyde(C-1inG6P)isreduced and the hydroxyl group at C-2 is oxidized to a ketone. This is D o accompaniedbyaprotonrearrangement.ConventionalPGIsof w EukaryaandBacteriafromavarietyofsourceshaveextensively n beeninvestigated,andthecrystalstructureshavebeenreported lo a forseveralorganisms(e.g.,pig,rabbit,human,andthebacterium d e Bacillusstearothermophilus).Theconservedaminoacidsproposed d to be involved in substrate binding and/or catalysis have been f r identified(forliterature,seereference74).PGIsfrommammalian o m sourcesexhibitthepropertiesofacytokineinvolvedincellmigra- h tionandproliferation(75,76).ConventionalPGIsarehomodi- t mericproteins((cid:6)120kDa),andeachsubunit((cid:6)60kDa)com- tp : prises two domains. The active center located at the domain // FIG2Ribbonrepresentationofthecrystalstructuresofthemonomersofthe interfaceismadeupofresiduesfrombothsubunits.Theconven- m differentsugarkinasesfoundinArchaea.(A)ADP-GLKfromPyr.furiosus m (PDBaccessionnumber1UA4)(59)asarepresentativeoftheADP-dependent tionalPGIsbelongtothePGIfamilywithinthePGIsuperfamilyof b sugarkinasefamilywithintheribokinasesuperfamilyalsocomprisingADP- proteins(SIS[sugarisomerase]domainsuperfamilyintheSCOP r. a PFKsaswellasthepromiscuousADP-GLK/PFKfromMca.jannaschii.(B) database)(45,77–80).InArchaea,homologsoftheseclassicalbac- s KDGKfromSul.solfataricus(PDBaccessionnumber2VAR)(122)asamem- terialandeukaryoticPGIshavebeenidentifiedonlyinhalophiles m beroftheribokinasesuperfamily,exhibitingthePFK-BfoldliketheATP- andsomemethanogens,andtheenzymefromMca.jannaschiihas .o dependentPFKspresentin,e.g.,Des.amylolyticusandAer.pernix.(C)HK r beencharacterized(81).Instead,PGIactivityinallotherArchaea g fromSul.tokodaii(PDBaccessionnumber2E2N)(49)asamemberofthe / actin-ATPasedomain-likesuperfamilyalsocomprisingROKhexokinasesand isassociatedwithtwootherproteinfamilies. o classicalHKsfromEukaryaandBacteria.(D)Classicalphosphofructokinase Phosphoglucoseisomerase/phosphomannoseisomerase.PGIs n fromE.coli(PDBaccessionnumber6PFK)(101)asamemberofthePFK-A havebeenpurifiedfromAer.pernix,Tpt.tenax,andPyb.aerophi- D family,towhichalsothePP-dependentPFKfromTpt.tenaxbelongs.All e i lumaswellasfromthemodifiedEDpathwayutilizerTpl.acido- c illustrationsofcrystalstructureswerepreparedbyusingthePymolMolecular e GraphicsSystem,version1.3(Schrödinger,LLC). philum(seebelow).Thecodingsequencesshowlowbutdetect- m ablesequencesimilaritiestotheclassicalPGIs,especiallyinthose b e residuesinvolvedincatalysis(82–84).However,incontrasttothe r unit(73).IncontrasttothePFK-Bkinases(seebelow),evidence G6P/F6P-specificconventionalPGIs,thecorrespondingenzymes 1 8 that a phosphoenzyme intermediate is involved in the haloar- showpromiscuousPGIandphosphomannoseisomerase(PMI) , chaeal KHK catalytic cycle has been reported and no antigenic activities,convertingbothG6Pandmannose6-phosphate(M6P) 2 0 cross-reactivityusingantibodiesraisedagainsthaloarchaealKHK toF6P.SimilartotheclassicalPGIs,thearchaealPGI/PMIsare 1 8 couldbedetectedwithmammalianKHKfromdifferentrattissues dimersandshowatwo-domainstructure.Eachdomainshowsan b (72).Thesefindingssuggestthatanovel,nonhomologousenzyme (cid:2)(cid:4)(cid:2)sandwichfold,whichisbuiltaroundaparallel(cid:4)sheet,five y is involved in fructose phosphorylation in halophilic Archaea. strandedintheN-terminaldomainandfourstrandedintheC- g However, the haloarchaeal KHK-encoding gene has not been terminal domain (Fig. 4). However, with a subunit size of (cid:6)35 ue s t FIG3Proposedreactionmechanismcatalyzedbysugarkinases,includingbase-mediatedprotonabstractionfromtheacceptorhydroxylgroupfollowedby nucleophilicattackonthe(cid:5)-phosphategroupofATP. 98 mmbr.asm.org MicrobiologyandMolecularBiologyReviews