Comparative Analysis of Pyruvate Kinases from the Hyperthermophilic Archaea Archaeoglobus ...

12 Pages·2003·0.57 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRY Vol.278,No.28,IssueofJuly11,pp.25417–25427,2003
©2003byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedinU.S.A.
Comparative Analysis of Pyruvate Kinases from the
Hyperthermophilic Archaea Archaeoglobus fulgidus,
Aeropyrum pernix, and Pyrobaculum aerophilum and the
Hyperthermophilic Bacterium Thermotoga maritima
UNUSUALREGULATORYPROPERTIESINHYPERTHERMOPHILICARCHAEA*
Receivedforpublication,October8,2002,andinrevisedform,March21,2003
Published,JBCPapersinPress,March21,2003,DOI10.1074/jbc.M210288200
UlrikeJohnsen,ThomasHansen,andPeterScho¨nheit‡
FromtheInstitutfu¨rAllgemeineMikrobiologie,Christian-Albrechts-Universita¨tKiel,AmBotanischenGarten1–9,
KielD-24118,Germany
Pyruvate kinases (PK, EC 2.7.1.40) from three hyper- mophilic archaea and in the hyperthermophilic bacterium
thermophilic archaea (Archaeoglobus fulgidus strain ThermotogarevealedthattheclassicEmbden-Meyerhof(EM)1
7324, Aeropyrum pernix, and Pyrobaculum aerophilum) pathway is operative only in Thermotoga, whereas in all ar-
andfromthehyperthermophilicbacteriumThermotoga chaea,theEMpathwayexistsinmodifiedversions.Themod-
maritimawerecomparedwithrespecttotheirthermo- ified EM pathways contain, e.g. unusual glucokinases (GLK)
philic,kinetic,andregulatoryproperties.PKsfromthe and 6-phosphofructokinases (PFK) such as ADP-dependent
archaea are 200-kDa homotetramers composed of 50- GLK and ADP-dependent PFK in Pyrococcus, Thermococcus,
kDa subunits. The enzymes required divalent cations, andArchaeoglobus;unusualATP-dependentarchaealGLKsof
Mg2(cid:1)andMn2(cid:1)beingmosteffective,butwereindepend-
theROK(Regulators,ORFs,Kinases)proteinfamily;non-reg-
ent of K(cid:1). Temperature optima for activity were 85°C
ulatory ATP-dependent PFKs in Desulfurococcus and Aeropy-
(A.fulgidus)andabove98°C(A.pernixandP.aerophi-
rum;andpyrophosphate-dependentPFKinThermoproteus.In
lum).ThePKswerehighlythermostableupto110°C(A.
addition,themodifiedEMpathwayscontainnovelenzymesof
pernix) and showed melting temperatures for thermal
glyceraldehyde3-phosphate(GAP)oxidationto3-phosphoglyc-
unfoldingat93°C(A.fulgidus)orabove98°C(A.pernix
erate, such as GAP:ferredoxin oxidoreductase and non-phos-
and P. aerophilum). All archaeal PKs exhibited sigmoi-
phorylativeglyceraldehyde-3-phosphatedehydrogenase,which
dal saturation kinetics with phosphoenolpyruvate
replace GAP dehydrogenase and phosphoglycerate kinase in
(PEP) and ADP indicating positive homotropic cooper-
ative response with both substrates. Classic hetero- theconventionalEMpathway(2–6).
tropic allosteric regulators of PKs from eukarya and An important regulatory principle of the carbon flux in the
bacteria,e.g.fructose1,6-bisphosphateorAMP,didnot classic EM pathway of eukarya and bacteria is the allosteric
affect PK activity of hyperthermophilic archaea, sug- regulationoftwokeyenzymes,ATP-dependentPFKandpyru-
gesting the absence of heterotropic allosteric regula- vatekinase.Bothenzymesareconsideredtocatalyzeirrevers-
tion. PK from the bacterium T. maritima is also a iblereactionsinvivoandhavebeenshowntobeallosterically
homotetramer of 50-kDa subunits. The enzyme was in- activatedorinhibitedbyintermediatesofmetabolismorbythe
dependent of K(cid:1) ions, had a temperature optimum of energy charge of the cell. To get insights into the role of allo-
80°C, was highly thermostable up to 90°C, and had a steric regulation of the modified EM pathways, the PFKs of
melting temperature above 98°C. The enzyme showed varioushyperthermophilicarchaeahavebeencharacterized.It
cooperativeresponsetoPEPandADP.Incontrasttoits wasfoundthatallarchaealPFKs(ADP-,ATP-,andpyrophos-
archaeal counterparts, the T. maritima enzyme exhib- phate-dependent) were not allosterically regulated by classic
itedtheclassicallostericresponsetotheactivatorAMP
effectors of ATP-PFKs of eukarya and bacteria, such as ADP
and to the inhibitor ATP. Sequences of hyperthermo-
andPEP(7–11).Thus,PFKsappearnottobeasiteofallosteric
philic PKs showed significant similarity to character-
control in the modified EM pathways of hyperthermophilic
ized PKs from bacteria and eukarya. Phylogenetic
archaea. In contrast, ATP-PFK from the hyperthermophilic
analysisofPKsequencesofallthreedomainsindicates
bacterium Thermotoga maritima shows the classic response
adistinctarchaealclusterthatincludesthePKfromthe
toward the allosteric effectors, it was activated by ADP and
hyperthermophilicbacteriumT.maritima.
inhibitedbyPEP(12).Thus,theATP-PFKofThermotogarep-
resentsasiteofallostericcontrolintheconventionalEMpath-
wayoperativeunderhyperthermophilicconditions.Toidentify
Hyperthermophilic prokaryotes, with an optimal growth
potential allosteric sites of the modified EM pathways of hy-
temperaturehigherthan80°C,areconsideredtorepresentthe
perthermophilicarchaea,westudiedtheregulatoryproperties
phylogeneticallymostancestralorganisms(1).Recentcompar-
ofpyruvatekinases,whichcatalyzetheirreversibleconversion
ativestudiesofthehexosedegradationpathwaysinhyperther-
*ThisworkwassupportedbygrantsoftheDeutscheForschungsge-
meinschaft and the Fonds der Chemischen Industrie. The costs of 1Theabbreviationsusedare:EM,Embden-Meyerhofpathway;GLK,
publicationofthisarticleweredefrayedinpartbythepaymentofpage glucokinases; PFK, 6-phosphofructokinases; GAP, glyceraldehyde
charges.Thisarticlemustthereforebeherebymarked“advertisement” 3-phosphate; FBP, fructose 1,6-bisphosphate; MES, 4-morpho-
inaccordancewith18U.S.C.Section1734solelytoindicatethisfact. lineethanesulfonic acid; ORF, open reading frame; Ni-NTA, nickel-
‡To whom correspondence should be addressed. Tel.: [phone]- nitrilotriacetic acid; CD, circular dichroism; aa, amino acid(s); PEP,
4328;Fax:[phone];E-mail:[e-mail]. phosphoenolpyruvate;LDH,L-lactatedehydrogenase.
This is an Open Access article under the CC BY license.
Thispaperisavailableonlineathttp://www.jbc.org 25417
25418 PKs from Hyperthermophilic Archaea and Bacteria
ofPEPtopyruvate,theterminalreactionofbothmodifiedand ruptedbypassingthroughaFrenchpressurecellat1.3(cid:2)108Pa.Cell
conventionalEMpathways. debrisandunbrokencellswereremovedbycentrifugationfor90minat
100,000(cid:2)gat4°C.
PKsarewellcharacterizedenzymesfrommanyeukaryaand
PurificationofPKfromA.fulgidus—The100,000(cid:2)gsupernatant
bacteria (13–15). The PKs are usually homotetrameric en- wasappliedtoaQ-SepharoseHiLoadcolumn(22(cid:2)5cm),whichhad
zymes of about 200 kDa composed of 50-kDa subunits; the beenequilibratedwithbufferA(50mMTris-HCl,pH9.0,1mMdithio-
enzymesrequiredivalentcationsforactivity;manyPKswere erythritol).ProteinwaselutedwithadecreasingpHgradientfrom9.0
showntobeactivatedbymonovalentcations,K(cid:1)orNH(cid:1).With to7.0inbufferAandfrompH7.0to6.5in50mMbis-Tris-propane,pH
4
a few exceptions, all PKs from eukarya and bacteria are allo- 6.5,containing1mMdithioerythritol.Fractionscontainingthehighest
PKactivitywerepooledand,afterpHexchange(pH9.0),appliedtoa
sterically regulated either by intermediates of sugar metabo-
Uno-Q5column(5ml),equilibratedwithbufferA.Proteinwasdesorbed
lism, usually sugar phosphates, or by adenosine nucleotides
withaNaClgradientfrom0to0.5MinbufferA.Fractionscontaining
reflectingtheenergychargeofthecells.MosteukaryalPKsare thehighestPKactivitywerepooledandconcentratedtoavolumeof1
allostericallyactivatedbyfructose1,6-bisphosphate(FBP).An mlbyultrafiltration.Theconcentratedproteinsolutionwasappliedto
unusual allosteric effector, fructose 2,6-bisphosphate, has re- aSuperdex200HiLoad16/60gelfiltrationcolumnequilibratedwith50
cently been reported for the PKs of the protozoa Leishmania mMMES,pH6.5,containing50mMNaCland1mMdithioerythritol.
Eluted PK-containing fractions were pooled and applied to a Uno-S1
mexicana and Trypanosoma brucei (16, 17). Several bacterial
column(1ml),equilibratedwith50mMacetate,pH5.3,containing1
PKs are activated by FBP, but the majority of bacterial PKs
mMdithioerythritol.ProteinwaselutedwithalinearNaClgradientof
showallostericactivationbyAMPandsugarmonophosphates 0to1M.Pureenzymewaselutedat0.25MNaCl.
(e.g. ribose 5-phosphate). Few bacteria, e.g. Escherichia coli PurificationofPKfromT.maritima—The100,000(cid:2)gsupernatant
and Salmonella typhimurium contain two PK isoenzymes be- wasappliedtoaQ-SepharoseHiLoadcolumn(22(cid:2)5cm),whichhad
ing activated either by FBP or by AMP (18–20). Few non- been equilibrated with 50 mM Tris-HCl, pH 7.0, containing 2 mM di-
allosteric PKs have also been described, e.g. M1 isoenzyme of thioerythritol. After washing the column with 50 mM piperazine, pH
6.4,containing2mMdithioerythritol(bufferB),proteinwaselutedwith
vertebrates and the dimeric PKs from Schizosaccharomyces
an increasing gradient from 0 to 2 M NaCl in buffer B. Fractions
pombe and Zymomonas mobilis (15, 21, 22). More than 140 containingthehighestPKactivity(0.15–0.3MNaCl)werepooledand
primarysequencesofeukaryaandbacteriaareknown.These applied to an SP-Sepharose column (75 ml) equilibrated with 50 mM
include putative PK homologs found in all available archaeal MES,pH5.5,containing2mMdithioerythritol.Proteinwasdesorbed
genomeswiththeexceptionofthemethanogensMethanopyrus with a pH gradient from pH 5.5 to 7.5 in 50 mM MES. Fractions
containingthehighestPKactivitywerepooledandconcentratedtoa
kandleri and Methanothermobacter thermoautotrophicus and
volumeof1mlbyultrafiltration(exclusionsize,20kDa).Theprotein
thehyperthermophilicsulfatereducerArchaeoglobusfulgidus
solution was applied to a Superdex 200 HiLoad 16/60 gel filtration
VC16(35).Interestingly,thecloselyrelatedstrainA.fulgidus columnequilibratedwith50mMTris-HCl,pH7.0,containing50mM
7324containshighpyruvatekinaseactivityaspartofamod- NaCl and 2 mM dithioerythritol. The eluted PK activity-containing
ifiedEMpathway(3).CrystalstructuresofPKsareavailable fractions were pooled and applied to a Uno-S1 column (1 ml) equili-
fortheenzymesfromcatandrabbitmuscle,yeast,andE.coli brated with 50 mM MES, pH 5.5, containing 2 mM dithioerythritol.
andofnon-allostericM1PKisoenzymesfromvertebrates(24– Proteinwaselutedwithalineargradientof0to1MNaCl.Fractions
containingthehighestPKactivitywerepooledandappliedtoaUno-Q1
27). The binding sites for PEP and for the allosteric activator
column(1ml)equilibratedwith50mMTris-HCl,pH8.0,containing1
FBPfromtheyeastPKwereidentified. mMdithioerythritol.ProteinwaselutedwithagradientofpH8.0to5.3
Todate,onlytwoPKsfromthedomainofarchaeahavebeen (50mMpiperazine,2mMdithioerythritol).Thefractioncontainingthe
biochemicallycharacterized,fromthehyperthermophileTher- highestPKactivitywaselutedatpH5.5,yieldingpureenzyme.
moproteustenaxandfromthemoderatethermophileThermo- CloningandFunctionalOverexpressionofORFTM0208Codingfor
PK of T. maritima and Purification of Recombinant Enzyme—ORF
plasma acidophilum. Both enzymes are homotetrameric pro-
TM0208 was amplified by PCR. The PCR product was cloned into
teins.FortheT.tenaxPKaresponsetoheterotropicallosteric
pET19b via two restriction sites (NdeI and BamHI) created with the
effectorswasnotfound,whereasthePKfromT.acidophilum primers 5(cid:3)-CGGGGTGAACATATGCGAAGTACAAAGAT-3(cid:3) and 5(cid:3)-
hasbeenshowntobeactivatedbyAMP(28,29). ATCTTCATAGGGATCCCCCCTCAATCCA-3(cid:3).ThevectorpET19b(pyk-
Inthiscommunicationweperformedacomparativestudyon TM0208)wastransformedintoE.coliBL21codonplus(DE3)-RILcells.
PKs,fromthreehyperthermophilicarchaea,thecrenarchaeota Forexpression,cellsweregrowninLuria-Bertanimediumat37°C.The
expressionwasstartedbyinducingthepromoterwithisopropyl-1-thio-
AeropyrumpernixandPyrobaculumaerophilumaswellasthe
(cid:1)-D-galactopyranoside.After3hoffurthergrowth,cellswereharvested
euryarchaeonA.fulgidusstrain7324andfromthehyperther-
bycentrifugation.ThepelletwassuspendedinbufferC(20mMTris-
mophilic bacterium T. maritima. The thermophilic, kinetic, HCl,pH8.2,containing0.3MNaCland4mMimidazole).Cellswere
and, in particular, the regulatory properties as well as the disrupted through a French pressure cell. After centrifugation
phylogeneticaffiliationofthePKswereanalyzed.Itwasfound (48,000 (cid:2) g, 4°C, 30 min), the supernatant was heat-precipitated at
thatallPKsfromhyperthermophilicarchaeaandbacteriaare 70°C for 30 min, followed by an additional centrifugation step
(100,000 (cid:2) g, 4°C, 60 min). The heat-precipitated supernatant was
homotetrameric proteins of extreme thermostability showing
appliedtoaNi-NTAcolumn(7ml)equilibratedwithbufferC.Protein
temperature optima up to 100°C for catalytic activity. An
waselutedwithincreasingimidazoleconcentrationfrom4to500mMin
unusual property of PKs from all hyperthermophilic archaea bufferC.Fractionscontainingthehighestenzymeactivitywerepooled
wastheabsenceofregulationbyclassicheterotropiceffectors. and concentrated to a volume of 1 ml by ultrafiltration. The protein
In contrast, the PK from the hyperthermophilic bacterium solution was applied to a Superdex 200 HiLoad 16/60 gel filtration
Thermotogashowedtheclassicresponsetoallostericeffectors. columnequilibratedwith50mMTris-HCl,pH7.5,containing150mM
Phylogenetic analysis of PK sequences of all three domains NaCland1mMdithioerythritol.Proteinwaseluted.Fractionscontain-
ingthehighestPKactivitywerepooledandappliedtoaUno-S1column
indicatesadistinctarchaealcluster.
(1ml)equilibratedwith50mMMES,pH5.5,containing1mMdithio-
erythritol.Proteinwaselutedwithagradientof0to1MNaCl.The
MATERIALSANDMETHODS fractioncontainingthePKactivitywasrecoveredat0.8MNaCl.Atthis
Growth of A. fulgidus and T. maritima and Preparation of Cell stagePKwasessentiallypure.
Extracts—A.fulgidusstrain7324andT.maritimaweregrownanaer- CloningandFunctionalOverexpressionofORFAPE0489Codingfor
obically in the presence of starch as described (3, 30–32). Cells were PK of A. pernix and Purification of Recombinant Enzyme—ORF
harvested in the late exponential growth phase. Cell extracts were APE0489 was amplified and cloned into pET19b via two restriction
preparedfrom80g(A.fulgidus)and60g(T.maritima)offrozencells, sites(NdeIandEcoR1)createdwiththeprimers5(cid:3)-TTAGAGAGGCT-
whichweresuspendedin150mlof50mMTris-HCl,pH7.0,containing GGCCTCATATGAGGGG-3(cid:3) and 5(cid:3)-GATAGGAATTCAGACAGGAGC-
10mMNaCland1mMdithioerythritolandin90mlof50mMTris-HCl, GGCTAG-3(cid:3).ThevectorpET19b(pyk-APE0489)wastransformedinto
pH7.0,containing2mMdithioerythritol,respectively.Cellsweredis- E.coliBL21codonplus(DE3)-RILcells.Theexpressionandcellhar-
PKs from Hyperthermophilic Archaea and Bacteria 25419
vestingwasperformedasdescribedabove.Thepelletwasresuspended 0.1-mm cuvettes and corrected for the signal of the solvent (20 mM
inbufferC.CellsweredisruptedbypassingthroughaFrenchpressure sodiumphosphate,pH7.0).Secondarystructureanalysisandassign-
cell. After centrifugation, the supernatant was heat-precipitated at ment to different secondary structure types were performed by the
77°Cfor30minandcentrifuged(100,000(cid:2)g,4°Cfor60min)again. experimentally established spectra-structure correlation using the
ThesupernatantwasappliedtoaNi-NTAcolumn(7ml)equilibrated VarselecoptionofDicroprot(33).Heat-inducedunfoldingofPKswas
withbufferC.Proteinwaselutedwithanimidazolegradientfrom4to analyzed in temperature gradient experiments. The protein samples
500mMinbufferC.FractionscontainingthehighestPKactivitywere weredialyzedagainst20mMsodiumphosphatebuffer,pH7.0,andthe
pooled,incubatedat100°Cfor15min,andcentrifuged.Atthisstage proteinconcentrationsweresetto100(cid:2)g/ml.Thetemperatureofthe
PKwasessentiallypure. samples was raised at a rate of 1°C per minute from 50 to 98°C.
CloningandFunctionalOverexpressionofORFPAE0819Codingfor Proteinunfoldingwasfollowedbytemperature-dependentchangeofa
PK of P. aerophilum and Purification of Recombinant Enzyme—ORF (cid:3)-helical ellipticity ((cid:4)) at 221 nm. The observed ellipticity ((cid:4) ) at a
obs
PAE0819wasamplifiedbyPCRandwasclonedintopET17bviatwo giventemperaturewascorrectedforthetemperature-dependentbase-
restrictionsites(NdeIandBamH1)createdwiththeprimers5(cid:3)-CAC- linetogive(cid:4)(cid:3) .Thefractionofunfoldedprotein(X )wascalculated
obs uf
TAAAGGGCGCGGACATATGAGCGCTC-3(cid:3) and 5(cid:3)-GTTGGGTACGC- usingthetemperature-correctedellipticitiesofthefolded((cid:4)(cid:3))andun-
f
CAGGATCCTCTTTTACCG-3(cid:3).ThevectorpET17b(pyk-PAE0819)was folded((cid:4)(cid:3) )states.Spectrawererecordedbeforeandaftereachtem-
uf
transformed into E. coli BL21 codon plus(DE3)-RIL cells. Expression peraturegradientexperimenttocharacterizethefoldedandunfolded
andcellharvestingwereperformedasdescribedabove.Thepelletwas states.Thefractionofunfoldedproteinwascalculatedaccordingtothe
resuspendedin50mMTris-HCl,pH8.5.Cellsweredisruptedbypass- equation,Xuf(cid:4)((cid:4)o(cid:3)bs (cid:5)(cid:4)f(cid:3))/((cid:4)u(cid:3)f (cid:5)(cid:4)f(cid:3)).
ingthroughaFrenchpressurecell.Aftercentrifugation,thesuperna- pHDependence,CationSpecificity,andEffectors—ThepHdepend-
tantwasheat-precipitatedat75°Cfor30minandcentrifuged.After enceoftheenzymeswasmeasuredbetween5.5and8.2at50°Cinthe
buffer exchange by ultrafiltration, the supernatant was applied to a continuous assay using either bis-Tris (pH 5.5–6.5), bis-Tris-propane
Uno-S5 column (5 ml) equilibrated with 50 mM piperazine, pH 5.3. (pH6.0–7.5),orTris-HCl(pH7.0–8.2)(A.fulgidus)andat65°Cusing
Protein was eluted with a gradient from 0 to 1 M NaCl. Fractions either piperazine (pH 5.5–6.1), bis-Tris (pH 6.1–6.5), or triethanol-
containingthehighestPKactivity(0.3–0.4MNaCl)werepooledand amine(pH6.5–7.5)(A.pernix,P.aerophilum,andT.maritima)(each
concentratedtoavolumeof1mlbyultrafiltration.Theproteinsolution 100mM).Cationspecificitieswereexaminedusingthestandardcon-
was applied to a Superdex 200 HiLoad 16/60 gel filtration column tinuous assay at 50°C (A.fulgidus) or 65°C (A. pernix, P. aerophi-
equilibrated with 50 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, lum, andT. maritima) as described above by replacing MgCl 2 for
andtheelutedPKwasessentiallypure. alternativedivalentcations(Mn2(cid:1),Co2(cid:1),Ca2(cid:1),Zn2(cid:1),Ni2(cid:1),orFe2(cid:1))
Enzyme Assays and Determination of Kinetic Parameters—The PK atequimolarconcentrations(0.1mM,1mM,and5mM).Thedepend-
activity was determined up to 65°C using a continuous assay in the enceonK(cid:1)andNH4(cid:1)wastestedusingconcentrationsupto100mM.
direction of pyruvate formation. It was ensured that the auxiliary The following classic allosteric effectors of PKs, fructose 1,6-
enzymewasnotrate-limiting.Oneunitofenzymeactivityisdefinedas bisphosphate, fructose 2,6-bisphosphate, AMP, L-alanine, ribose
1(cid:2)molofproductformedperminute.Theassaymixturecontainedfor 5-phosphate,glucose6-phosphate,fructose6-phosphate,citrate,and
A. fulgidus and T. maritima 100 mM triethanolamine, pH 7.0, 1 mM erythrose4-phosphate(concentrationrangebetween10(cid:2)Mand5mM)
PEP,2mMADP,5mMMgCl2,0.3mMNADH,and1unitofLDH,and, weretestedat65°Cusingthecontinuousassayasdescribedabove
forA.pernixandP.aerophilum100mMbis-Tris,pH6.2,1mMPEP,2 with both PEP and ADP concentrations near their S0.5 values: A.
mMADP,5mMMgCl2,0.3mMNADH,and1unitofLDH.Theformation fulgidus,0.4mMMgCl2,0.4mMADP,and0.2mMPEP;P.aerophi-
ofpyruvatefrom65to98°Cwasmeasuredbyusingadiscontinuous lum, 7.5 mM MgCl2, 1.5 mM ADP, and 0.5 mM PEP; A. pernix, 1 M
assay.Thestandardassaymixture(250(cid:2)l)contained100mMTris-Cl, MgCl2,0.5mMADP,and0.3mMPEP.InthecaseofA.pernixPKthe
1–5 mM PEP, 2 mM ADP, 5–10 mM MgCl2. After preincubation, the effectors were preincubated with the protein at the respective tem-
reactionwasstartedwithanaliquotofPK,incubatedfor15–120s,and perature.TheassayforT.maritimaPKcontained2.5mMADP,2.5
stoppedbyrapidadditionof750(cid:2)lofice-coldbuffer(100mMTris-HCl, mM MgCl2, and 0.3 mM PEP in 0.1 M MES, pH 6.5 (65°C). When
pH7.0,0.6mMNADH,0.5unitofLDH);theamountofpyruvateformed effectorsweretested,thesubstratesADPandPEPwereusedatthe
wasquantifiedbyfollowingtheoxidationofNADHat365nm.Kinetic highestpurityavailable.
parameters of PKs were determined at 65°C using the continuous Sequence Handling—Sequence alignments were constructed with
assay(seeabove).Sixtoeightdifferentconcentrationsofthesubstrates theNeighbor-joiningmethodofClustalX(34)usingtheGONNETma-
PEPandADPwereused.Theassaymixturescontained0.3mMNADH trix. Phylogenetic trees were constructed using both the Neighbor-
and1unitofLDHforallPKsand,specifically,asfollows:A.pernix,0.1 joiningoptionofClustalXaswellastheMaximum-likelihoodmethodof
Mbis-Tris,pH6.2,and0–1mM(ADP/2MgCl2),0.5mMPEPor0–1mM PROML(Phylip,version3.6).Confidencelimitswereestimatedby100
PEP,0.5mMADP,1mMMgCl2;A.fulgidus,0.1Mtriethanolamine,pH bootstrappingreplicates.
7,and0–3mMADP/MgCl2,0.4mMPEPor0–2mMPEP,0.2mMADP, SourcesofOrganisms—A.fulgidusstrain7324(DSM8774),A.per-
1 mM MgCl2; P. aerophilum, 0.1 M bis-Tris, pH 6.2, and 0–2.5 mM nix(DSM11879),P.aerophilum(DSM7523),andT.maritima(DSM
ADP/12.5MgCl2,1mMPEPor0–1mMPEP,1.5mMADP,and7.5mM 3109)wereobtainedfromtheDeutscheSammlungvonMikroorganis-
MgCl2;andT.maritima,0.1MMES,pH6.5,and0–3mMADP/MgCl2, menundZellkulturen(Braunschweig,Germany).
5mMPEPor0–3mMPEP,2.5mMADP/MgCl2.Kineticconstantsand
standarderrorsareobtainedfrombest-fitcurves.Thedatapointsgiven RESULTS
inthefiguresareoriginalmeasurementsofoneexperiment;thecurves PyruvateKinasesfromtheHyperthermophilicArchaea
drawnrepresentfitstoasigmoidalmodelorahyperbolicmodel(Fig.4, A.pernix,P.aerophilum,andA.fulgidusStrain7324
ThermotogaPKactivityinthepresenceofAMP)accordingtonon-linear
regressionanalysisusingtheMicrocalTMOriginTMsoftwareversion5.0. In the genomes of the hyperthermophilic crenarchaeota A.
InthedeterminationoftheHillcoefficients,thebest-fitlinesgenerated pernixandP.aerophilumORFAPE0489andPAE0819,respec-
via linear regression analysis by the same software are shown (e.g. tively, were annotated as putative pyk genes coding for pyru-
Fig.1B).
vate kinase. To prove their coding function, the ORFs were
TemperatureDependenceandThermalStability—Thetemperature
clonedandfunctionallyexpressedinE.coli.Therecombinant
dependenceofPKactivitieswasmeasuredbetween20and98°C,using
thediscontinuousassay,in100mMtriethanolamine,pH7.0(A.fulgi- proteins were characterized. In the genome of A. fulgidus
dus), or 100 mM bis-Tris, pH 6.2 (A. pernix, P. aerophilum, and strainVC16,nopykhomologousgenewasidentified.Because
T.maritima)eachcontaining1mMPEP,2mMADP,and5mMMgCl2. the closely related strain A. fulgidus 7324 has been shown to
LongtermthermostabilityofPKs(0.5(cid:2)gin30(cid:2)lof100mMtrietha- contain high PK activity after growth on starch (3), PK was
nolamine,pH7.0(A.fulgidus),1.5(cid:2)gin30(cid:2)lof100mMbis-Tris,pH6.2 purifiedandcharacterizedfromthisArchaeoglobusstrain.
(A.pernix),1.4(cid:2)gin30(cid:2)lof100mMsodiumphosphatebuffer,pH7.0
(P.aerophilum),and1(cid:2)gin30(cid:2)lof100mMtriethanolamine,pH7.0
PKfromA.fulgidusStrain7324
(T.maritima),eachattherespectivetemperature)weretestedinsealed
vials,whichwereincubatedattemperaturesbetween70and110°Cup
ExtractsofA.fulgidusgrownonstarchascarbonandenergy
to120min.Thevialswerecooledfor10min,andtheremainingactivity
source contained PK activity (0.13 unit/mg, 50°C), which is
wastestedinacontinuousassay.
Circular Dichroism Spectroscopy—CD spectroscopy analyses were about 5-fold higher as compared with PK activity of lactate
performedonaJascoJ-715CDspectrometer.Spectrawererecordedin growncells(0.02–0.04unit/mg)indicatingacatabolicfunction
25420 PKs from Hyperthermophilic Archaea and Bacteria
TABLE I
MolecularandkineticpropertiesofpurifiedrecombinantpyruvatekinasesfromA.pernixandP.aerophilumandof
purifiedpyruvatekinasefromA.fulgidus
Kineticconstantsweremeasuredat65°C,andstandarderrorsaregiven(see“MaterialandMethods”).
A.pernix P.aerophilum A.fulgidus
Apparentmolecularmassofenzyme(kDa) 207(cid:6)18 205(cid:6)7 203(cid:6)14
Apparentmolecularmassofsubunits(kDa) 51(cid:6)3 48(cid:6)3 49(cid:6)3
Oligomericstructure (cid:3) (cid:3) (cid:3)
4 4 4
pHoptimum 6.1 6.0 6.6
T (°C) (cid:7)98 (cid:7)98 93
Tm (°C) (cid:7)95 (cid:7)98 85
opt
Arrheniusactivationenergy(kJ/mol) 65a 55b 56c
ApparentV (units/mg) 53(cid:6)1 46(cid:6)1 1000(cid:6)15
AHpilplacroeenffticSie0m.n5atxfo(hr)ADP(mM) 20..1206(cid:6)(cid:6)00..1073 21..6391(cid:6)(cid:6)00..2054 20..1410(cid:6)(cid:6)00..105237
HApilplacroeenffticSie0.n5tfo(hr)PEP(mM) 10..5130(cid:6)(cid:6)00..1033 20..8421(cid:6)(cid:6)00..3001 10..8205(cid:6)(cid:6)00..1013
LinearpartoftheArrheniusplot:a35–95°C,b20–98°C,c20–80°C.
of the enzyme during sugar degradation (3). PK was purified kineticswithrespecttothesubstratesPEPandADP,indicat-
from starch-grown cells to homogeneity using four chromato- ing a positive homotropic cooperative response to both sub-
graphic steps. The enzyme was purified about 1200-fold to a strates(Fig.1andTableI).PKactivitiesofallarchaearequire
specificactivityof1000units/mgat80°Cwithayieldof7%. divalentcationsandwerenotdependentonpotassium.
A.fulgidusPK—Thepurifiedenzymehadaspecificactivity
MolecularCompositionandN-terminal
of1000units/mg.TheapparentS valuesforADPandPEP,
AminoAcidSequence 0.5
calculated from sigmoidal fit, were 0.4 and 0.25 mM, and the
Thenativeenzymehadanapparentmolecularmassof203 corresponding Hill coefficients were 2.1 and 1.8, respectively.
kDaandshowedone49-kDabandonSDS-PAGEindicatinga PKactivityrequireddivalentcations.RatedependenceofMg2(cid:1)
homotetrameric ((cid:3)) structure of the enzyme (Table I). The showed sigmoidal kinetics, indicating cooperative response of
4
N-terminal amino acid sequence (20 amino acids, aa) of the PKtothiscation.AnapparentS0.5valueof0.7mMandaHill
subunit was determined: MQLPSHKTKIIATIGPASRQ. An coefficient of 1.3 were calculated. Alternative divalent cations
alignmentoftheN-terminalaminoacidsequencefromA.fulgi- weretestedatconcentrationsof0.1mM,1mM,and5mM.For
dus PK with putative PKs from hyperthermophilic archaea mostcations(exceptFe2(cid:1))1mMconcentrationwasnotinhib-
showed the highest degree of identity with hypothetical PK itory. At 1 mM concentration the highest PK activity was ob-
from Thermococcus litoralis (18 aa identical) and Pyrococcus served with Mg2(cid:1) (100% (cid:4) 1060 units/mg at 50°C), which
furiosus (15 aa identical). Surprisingly, using the N-terminal could be replaced by Cu2(cid:1) (86%) and Mn2(cid:1) (63%) and less
sequenceofPKfromA.fulgidusstrain7234,noORFcouldbe efficiently by Ni2(cid:1) (2%), Ca2(cid:1) (7%), and Zn2(cid:1) (6.5%). The pH
identifiedinthecompletesequencedgenomeofcloselyrelated optimum was at pH 6.6; 50% of activity was found at pH 5.5
A. fulgidus VC 16 (35), thus confirming the absence of a pyk and7.5.
homologousgeneintheA.fulgidustypestrain. P.aerophilumPK—ThespecificactivityofPKwas46units/
FunctionalOverexpressionofORFPAE0819and mg. The apparent S0.5 values for ADP and PEP were 1.3 mM
and 0.4 mM; the calculated Hill coefficients were 2.7 and 2.8,
APE0489EncodingPKsfromtheArchaeaP.aerophilum respectively(Fig.1).ThehighestactivitywasfoundwithMn2(cid:1)
andA.pernixandPurificationoftheRecombinantPKs (100%(cid:4)200units/mgat65°C)andCo2(cid:1)(80%)(eachat1mM
ORFPAE0819contains1386bpcodingforapolypeptideof concentration). Remarkably, the enzyme did not show signifi-
461aminoacidswithacalculatedmolecularmassof50.3kDa. cantactivity(about1%)withMg2(cid:1)(1mM).Therelativeactivity
The ORF was cloned and expressed in E. coli. The PK was with Mg2(cid:1), however, was about 58% as compared with that
purified from E. coli by heat treatment and two chromato- with Mn2(cid:1) (100% (cid:4) 0.75 unit/mg), when the cations were
graphicstepstoaspecificactivityof46units/mgat65°C.ORF testedat0.1mMconcentration.Noactivitywasobservedwith
APE0489,annotatedasaputativepykgeneinA.pernix,con- Ca2(cid:1)andZn2(cid:1).ThepHoptimumwasatpH6.
tains1374bpcodingforapolypeptideof458aminoacidswith A.pernixPK—ThespecificactivityofPKwas53units/mg.
acalculatedmolecularmassof50.5kDa.TheORFwascloned TheapparentS0.5valuesforADPandPEPwere0.26and0.1
and expressed in E. coli. The His-tagged PK was purified by mM, and the calculated Hill coefficients were 2.1 and 1.5,
heat treatment, chromatography on Ni-NTA agarose, and a respectively. PK activity required divalent cations. Highest
secondheattreatmentstepat100°Ctoaspecificactivityof53 activities (1 mM cation) were determined with Mg2(cid:1) (100%),
units/mgat65°C.ThepurifiedPKsfromP.aerophilumandA. Co2(cid:1)(170%),andMn2(cid:1)(160%).Ca2(cid:1)(14%),Zn2(cid:1)(11%),and
pernix each showed apparent molecular masses of about 200 Ni2(cid:1)(14%)werelessefficient.WithMg2(cid:1)theenzymeshowed
kDa;SDS-PAGErevealedonesubuniteachwithapparentmo- cooperativebindingandrevealedaS0.5of0.7mMandaHill
lecular masses of 48 and 51 kDa, respectively, indicating a coefficient of 1.4. The pH optimum of the enzyme was at
homotetramericstructureofbotharchaealPKs(TableI). pH 6.1.
CatalyticPropertiesofPKsfrom TemperatureOptimumandThermostabilityof
HyperthermophilicArchaea PKsfromHyperthermophilicArchaea
Thecatalytic,thermophilic,andregulatorypropertiesofthe A.fulgidusPK—PKactivityshowedatemperatureoptimum
PKsfromA.fulgidus,P.aerophilum,andA.pernixwereana- at 85°C. The enzyme was highly thermostable, did not lose
lyzed(TableI).AllarchaealPKsshowedasigmoidalsaturation significantactivityuponincubationat70°Cfor120min,and
PKs from Hyperthermophilic Archaea and Bacteria 25421
P. aerophilum PK—PK activity showed an optimum at
higher than 98°C, the highest possible temperature. The en-
zyme showed high stability against heat inactivation with a
half-lifeof220minat100°C.
A. pernix PK—PK activity showed a temperature optimum
higher than 95°C (Fig. 2, A and B). The enzyme showed the
highestthermostabililyofallarchaealPKs.Theenzymedidnot
lose activity upon incubation for 120 min at 100°C. Even at
110°C the PK showed a half-life of about 30 min (Fig. 2C).
Additionof(NH4)2SO4,NaCl,orKCl(each1M)didnotstabilize
PKactivityagainstheatinactivationat110°C.
ThermostabilityofPKsfromHyperthermophilicArchaea
AnalyzedbyCDSpectroscopy
ThehighstabilityofPKsagainstheatinactivationwasfur-
thersupportedbyfollowingheat-inducedunfoldingofthepro-
teinsupto98°CbyCDspectroscopyat221nm.Unfoldingwas
observedonlyforPKofA.fulgidusshowingameltingtemper-
ature(T )of93°C.NounfoldingwasdetectedwithPKsfrom
m
P.aerophilumandA.pernixuptotemperaturesof98°C(Fig.
3),indicatingmeltingtemperatureshigherthan100°C.Thisis
in accordance to the higher temperature optima for catalytic
activityandthethermostabilitiesofthelatterPKsascompared
withtheA.fulgidusPK.
EffectofAllostericEffectorsonPKsfrom
HyperthermophilicArchaea
The effect of classic positive allosteric effectors for PKs of
most eukarya and bacteria, such as FBP and AMP, were
tested at 65°C on PK activity from A. fulgidus, P. aerophi-
lum,andA.pernix(see“MaterialsandMethods”).Therewas
noactivationeffectobservedbyanyoftheligandstestedwith
the PKs studied. ATP has been reported to be an allosteric
inhibitor of several PKs from eukarya and bacteria, inhibi-
tion being reversed by positive allosteric effectors FBP or
AMP. ATP (1 mM) inhibited activities of archaeal PKs, e.g.
about 55% in A. fulgidus PK (at 0.3 mM PEP, 0.2 mM ADP).
Inhibition could not be reversed by the addition of FBP or
AMP(1mMeach).However,ATP-inducedinhibitioncouldbe
reversed up to 90%, by increasing the PEP concentration
from 0.3 mM to 1 mM or by the addition of 1 mM ADP,
indicating competitive inhibition. Inhibition of activity by
ATP, competitive to ADP and PEP, has also been described
for other PKs (36).
Theapparentabsenceofallostericregulationbyheterotropic
compoundsofthePKsfromhyperthermophilicarchaeamight
beduetotheirhyperthermophilicnatureand/orduetoasyet
unknown different regulatory mechanism of the modified EM
pathways of archaea. Thus, for comparison we characterized
the PK from the hyperthermophilic bacterium T. maritima,
whichusestheconventionalEMpathwayforglucosedegrada-
tion.Boththenativeenzymeand,forstructuralandfunctional
analysis,therecombinantPKwereanalyzed.
PyruvateKinasefromtheHyperthermophilicBacterium
T.maritima
CellextractsofT.maritimagrownonstarchascarbonand
FIG.1.RatedependenceofpyruvatekinasefromP.aerophi- energysourcecontaineda5-foldhigherPKactivity(0.13unit/
lumonsubstrateconcentrations.A,PEPsaturationcurve;B,Hill
mg,50°C),ascomparedwithPKactivityofcells(0.02unit/mg)
plotofthesamedata;andC,ADPsaturationcurve.
grownonyeastextractindicatingtheinductionoftheenzyme
during sugar catabolism. PK was purified from starch-grown
showedahalf-lifeofabout20minat90°C.At100°Canalmost cells to homogeneity in five chromatographic steps. The en-
completelossofactivitywasobservedafter7min.Additionof zymewaspurifiedabout2000-foldtoaspecificactivityof320
1M(NH4)2SO4,ratherthanNaClorKCl(1Meach),effectively units/mgat70°Cwithayieldof7%.Thenativeenzymehada
stabilized PK against heat inactivation at 100°C, retaining molecularmassof194kDaandwascomposedof51-kDasub-
about50%residualactivityafterincubationat120min. units indicating a homotetrameric structure (Table II). The
25422 PKs from Hyperthermophilic Archaea and Bacteria
FIG.3. Thermal induced unfolding of pyruvate kinases from
the archaea A. fulgidus, A. pernix, and P. aerophilum and
fromthebacteriumT.maritimameasuredbyCDspectroscopy
at221nm.
FunctionalOverexpressionofTM0208EncodingPKfrom
T.maritimaandPurificationoftheEnzyme
ORF TM0208 contains 1398 bp coding for a polypeptide of
466aminoacidswithacalculatedmolecularmassof51.9kDa.
The ORF was cloned and expressed in E. coli. The PK was
purifiedbyheattreatmentinthreechromatographicsteps.The
His-tagged PK showed a molecular mass of 210 kDa and a
subunits size of 56 kDa on SDS-PAGE indicating a homotet-
ramericstructure.
Catalytic,Thermophilic,andRegulatoryPropertiesof
NativeandRecombinantPKfromT.maritima
TheapparentV values(at65°C)forpyruvateformation
max
ofthenativeandtherecombinantPKwere320and580units/
mg, respectively. The lower activity of the native enzyme is
probably due to the damage during time-consuming purifica-
tionprocedure(2000-foldpurificationinfivechromatographic
steps).Both,nativeandrecombinantPKwerealmostidentical
withrespecttothefollowingproperties.Theenzymesshowed
positively cooperative response to both ADP and PEP with
apparentS0.5valuesof1.3and0.3mM;thecorrespondingHill
coefficientswere2.9and2.1,respectively(TableIIandFig.4).
ThepHoptimumwasnear6.0;30%oftheactivitywasfoundat
pH5.5and7.0.PKactivityrequireddivalentcations;at1mM
concentration, Mg2(cid:1) (100%) could be efficiently replaced by
Co2(cid:1)(120%)andMn2(cid:1)(35%)ratherthanbyCa2(cid:1)(3%),Zn2(cid:1)
(2.5%), Ni2(cid:1) (3%), or Fe2(cid:1) (1.5%). Mg2(cid:1) showed cooperative
responsetotheenzymewithaS0.5of1mMandaHillcoefficient
of 2.3. PK activity was not dependent on monovalent cations,
such as K(cid:1) and NH4(cid:1). Addition of both KCl or NH4Cl (40 mM
each) resulted in a decrease of PK activity (recombinant) by
50–60%.
TemperatureOptimumandStability
PK activity (recombinant) showed an temperature opti-
FIG.2.Effectoftemperatureonthespecificactivityandther-
mostability of pyruvate kinase from A. pernix. A, temperature mumat80°C.BothnativeandrecombinantPKshowedhigh
dependenceofthespecificactivity;B,Arrheniusplotofthesamedata; thermostability up to 85°C; even at 100°C the enzyme
andC,thermostabilityat100°C((cid:1))andat110°C(f).100%activity showed a half-life of about 20 min, but an almost complete
correspondedto50units/mg.
loss of activity was observed after 120 min. Addition of
(NH4)2SO4,ratherthanNaClorKCl(each1M),stabilizedPK
N-terminal amino acid sequence of the subunit (MRST- against heat inactivation at 100°C, retaining about 40%
KIVCTVGPRTD) was identical to the deduced N-terminal se- residual activity after 120-min incubation. Thermal unfold-
quenceoftheORFTM0208,whichisannotatedasaputative ingofPK,asmeasuredbyCDspectroscopy,wasnotobserved
pykgeneencodingpyruvatekinase. up to 98°C (Fig. 3).
PKs from Hyperthermophilic Archaea and Bacteria 25423
TABLE II
MolecularandkineticpropertiesofthepurifiednativeandrecombinantpyruvatekinasefromT.maritima
Kineticconstantsweremeasuredat65°C,andstandarderrorsaregiven(see“MaterialandMethods”).
Nativeenzyme Recombinantenzyme
Apparentmolecularmassofenzyme(kDa) 194(cid:6)14 190(cid:6)7
Apparentmolecularmassofsubunits(kDa) 51(cid:6)3 56(cid:6)3
Oligomericstructure (cid:3) (cid:3)
4 4
pHoptimum 6.0 5.9
T (°C) NDa (cid:7)98
m
T (°C) ND 80
opt
Arrheniusactivationenergy(kJ/mol,30–70°C) 61.4 55.6
ApparentV (units/mg) 320(cid:6)7 578(cid:6)10
max
ADPsaturation
AHpilplacroeenffticSie0.n5t(m(hM)) 12..3855(cid:6)(cid:6)00..0275 12..3811(cid:6)(cid:6)00..1174
PEPsaturation
(cid:5)Effector HApilplacroeenffticSie0.n5t(m(hM)) 01..2965(cid:6)(cid:6)00..0137 02..2230(cid:6)(cid:6)00..0065
(cid:1)AMPb AHpilplacroeenffticSie0.n5t(m(hM)) 01..0392(cid:6)(cid:6)00..0069 01..0080(cid:6)(cid:6)00..0150
(cid:1)ATPb HApilplacroeenffticSie0.n5t(m(hM)) NNDD 02..59(cid:6)(cid:6)00..0143
aND,notdetermined.
bConcentrationsofeffectorswere1mM.
FIG.5.CDspectraofpyruvatekinasesfromT.maritima(——)
andP.aerophilum((cid:1)(cid:1)(cid:1)(cid:1)).
mainedalmostconstant.Thus,e.g.ataPEPconcentrationof
0.1mM,AMPactivatesPKactivityupto10-fold.Conversely,
theadditionofATPresultedinanallostericinhibitionofPK
onFPIGE.P4.cRoantceednetrpaetniodnenicnetohfeppyrreusveantceekainndasaebfsreonmceTo.fmeaffreitcitmora. activity by increasing S0.5 from 0.23 to 0.5 mM; the Hill
Noeffector(f),1mMAMP((cid:1)),and1mMATP(Œ). coefficient increased to 2.9, and Vmax was reduced to 70%.
InhibitionbyATPwascompletelyreversedbytheadditionof
theactivatorAMP(1mM).Thus,incontrasttothePKsfrom
EffectofAllostericEffectorsonPKActivity hyperthermophilicarchaea,bothAMPandATPexertedtheir
classicallostericeffectstowardthehyperthermophilicPKof
Theeffectofclassicallostericactivators,suchasAMPand
the bacterium T. maritima.
FBP, and of the allosteric inhibitor ATP was tested on PK
activityat65°C.Theratedependenceofenzymeactivityon
CDSpectraofHyperthermophilicPKsfromtheArchaeon
increasingPEPconcentrationsinthepresenceofAMPandof
P.aerophilumandtheBacteriumT.maritima
ATP is shown in Fig. 4. In the absence of effectors, rate
dependence of PK (recombinant) showed sigmoidal kinetics To get information about the secondary structure of PKs
with an S value of 0.23 and a Hill coefficient of 2.2. Addi- fromhyperthermophiles,CDspectrawererecordedforPKfrom
0.5
tionofAMP,ratherthanofFBP,allostericallyactivatesthe Pyrobaculum and Thermotoga. The spectra of both PKs were
enzyme: rate dependence on PEP changed from a sigmoidal almostsuperimposable(Fig.5).ForthePKfromP.aerophilum
kinetics to a hyperbolic, Michaelis-Menten kinetics, paral- an(cid:3)-helicalcontentof36%anda(cid:1)-sheetcontentof25%were
leledbythedecreaseinS0.5forPEPfrom0.23mMtoaKmof estimated,whichcloselymatchthesecondarystructurepredic-
0.08 mM; the Hill coefficient decreased to 1.0, and Vmax re- tions (36% (cid:3)-helical and 26% (cid:1)-sheet) for both enzymes. The
25424 PKs from Hyperthermophilic Archaea and Bacteria
FIG.6. Multiple sequence alignment of amino acid sequences of pyruvate kinases from eukarya, bacteria, and archaea. The
alignmentwasgeneratedwithClustalX.Conservedresiduesthathavebeenproposedtobeindispensableforcatalyticactivityasdeducedfromthe
yeast x-ray structure (26) are indicated by asterisks. The arrow indicates conserved Glu residue essential for K(cid:1) dependence. The consensus
patternisindicatedbyabox.ThepredictedsecondarystructureoftheP.aerophilumpyruvatekinaseisshownabovethesequences.Foraccession
numbersseeFig.7.
secondarystructureestimationswerecomparabletothosede- ity described so far. For example, PK from P. aerophilum
rivedfromthex-raystructuresofyeast(38%(cid:3)-helicaland12% (optimalgrowthtemperature,100°C)showedatemperature
(cid:1)-sheet)andE.coliPK(38%(cid:3)-helicaland21%(cid:1)-sheet). optimum higher than 98°C and was heat-resistant up to
100°C for 2 h. In addition, thermal unfolding experiments
DISCUSSION
revealed extremely high melting temperatures of the PKs
Molecular and Thermophilic Properties—The PKs from the
near or above 100°C. For comparison, PK from the extreme
hyperthermophiles were characterized as homotetramers of
thermophilic bacterium Thermus was completely heat-
about 200 kDa composed of 50-kDa subunits, which is a com-
monfeatureofPKsfrombacteriaandeukarya,andofthetwo inactivated in less than 10 min at 100°C (37).
archaeal PKs characterized so far, from the crenarchaeon Kinetic and Regulatory Properties—All hyperthermophilic
T. tenax and the euryarchaeon T. acidophilum (28, 29). In PKsrequiredivalentcationsforactivity,acommonpropertyof
accordance with the optimal growth temperatures of the re- allcharacterizedPKs;Mn2(cid:1),Mg2(cid:1),orCo2(cid:1)beingmosteffec-
spective organism, the hyperthermophilic PKs of our study tive. The PKs from the hyperthermophilic archaea and from
showedthehighest temperature optimum and thermostabil- Thermotogawerenotdependentonmonovalentcationssuchas
PKs from Hyperthermophilic Archaea and Bacteria 25425
FIG.7.Phylogeneticrelationshipsofpyruvatekinasesfrombacteria,eukarya,andarchaea.Thenumbersatthenodesarebootstrap-
ping values according to neighbor-joining (values on top) and maximum-likelihood (values beneath). NCBI accession numbers or SwissProt
identifiers: A. pernix, BAA79454; Bac.lic., Bacillus licheniformis KPYK_BACLI; Bac.ste., Bacillus stearothermophilus S29783; Cor.glu., C.
glutamicumKPYK_CORGL;Dei.rad.,DeinococcusradioduransAAF12171;E.coli1,KPY1_ECOLI;E.coli2,KPY2_ECOLI;Cat,KPY1_FELCA;
Hae.inf.,HaemophilusinfluenzaeKPYK_HAEIN;Human,KPY2_HUMAN;Hyd.the.,HydrogenophilusthermoluteolusBAA95686;Lac.del.,Lac-
tobacillusdelbrueckiiKPYK_LACDE;Lac.lac.,LactococcuslactisB40620;Lei.mex.,L.mexicanaKPYK_LEIME;M.jannaschii,Methanococcus
jannaschiiD64313;M.acetivorans,Methanosarcinaacetivoransstr.C2AAAM07241;M.mazei,MethanosarcinamazeiGoe1AAM30411;Rabbit,
KPY1_RABIT;P.aerophilum,AAL63053;Pyrococcusabyssi,CAB50316;P.furiosus,AAL81312;P.hor.,PyrococcushorikoshiiF71171;yeast1,
KPY1_YEAST;yeast2,KPY2_YEAST;Sal.typ.1,S.typhimuriumLT2AAL20302;Sal.typ.2,S.typhimuriumLT2AAL20804;Str.the.,Streptococ-
cus thermophilus AAF25804; Str.coe., Streptomyces coelicolor T35759; Str.coe., A3 CAB70653; Sulfolobus solfataricus, AAK41255; Sulfolobus
tokodaii, BAB66695; T. acidophilum, KPYK_THEAC; Thermoplasma volcanium, BAB60191; T. tenax, AAF06820; T. maritima, AAD35300;
Try.bru.,T.bruceibruceiKPY2_TRYBB.
K(cid:1) and NH(cid:1). K(cid:1)-independent PKs have been reported for 1,6-bisphosphate(FBP),othersugarphosphates,orAMP.Also
4
E. coli, Corynebacterium glutamicum, and Z. mobilis and for fructose2,6-bisphosphate,theallostericactivatoroftheLeish-
the archaeon T. tenax (18, 22, 29, 38). PK sequences contain maniaPK(16),didnothaveaneffect.Thus,thehyperthermo-
highly conserved K(cid:1) sites (residues 48–52 and 79–88 of the philic archaeal PKs differ from PKs of bacteria and eukarya,
Pyrobaculum PK), including a glutamate in K(cid:1)-stimulated whichareallostericallyactivatedbythesecompounds.ATPhas
PKs, e.g. Glu-89 of the yeast PK (Fig. 6). This glutamate is beendescribedtobeanallostericinhibitorofvariouseukaryal
substitutedinpotassium-independentPKs.Inaccordancewith andbacterialPKs,withinhibitionsbeingreversedbytheacti-
thelackofthedependenceonpotassium,allknownhyperther- vatorFBPorAMP(39).Incontrast,theATPinhibitionofPKs
mophilic PKs have substituted glutamate at the equivalent fromhyperthermophilicarchaeainthisstudyiscompetitiveto
position (e.g. by Arg, Lys, or Ser). The PK from the moderate substrates PEP and ADP and could not be reversed by AMP
thermophilic archaeon T. acidophilum, which contains this orFBP.
glutamate,wasdescribedtobedependentonpotassium(28). An apparent absence of heterotropic allosteric regulation
AllhyperthermophilicPKsshowedasigmoidalratedepend- has also been reported for the PK of the hyperthermophilic
enceforthesubstratesPEPandADPandforMg2(cid:1),indicating archaeon T. tenax (29). However, the PK from the archaeon
positively homotropic cooperative response to substrates and T.acidophilumhasbeendescribedtobeactivatedbyAMP(28),
cations.Cooperativesubstratebindinghasalsobeendescribed indicatingthatthereducedregulatorycapacityisprobablynot
forafewPKsfromeukaryaandbacteria.However,manyPKs, ageneralfeatureofallarchaealPKs.Incontrasttothehyper-
including the PK of the hyperthermophilic archaeon T. tenax thermophilic PKs, the PK from the hyperthermophilic bacte-
(29),havebeenreportedtoshowhyperbolicratedependenceon riumT.maritimashowedtheclassicallostericresponsetothe
ADP, suggesting a different conformational response of these allostericregulatorsofbacteria.Itwasallostericallyactivated
PKstoADPbinding. by AMP and inhibited by ATP. Inhibition of ATP could be
An important result of this study is the apparent lack of reversedbyAMP.
allosteric regulation of the PKs from the hyperthermophilic Thereasonsfortheabsenceofclassicheterotropicregulation
archaea by classic heterotropic compounds such as fructose of PKs in hyperthermophilic archaea are not understood. A
25426 PKs from Hyperthermophilic Archaea and Bacteria
specific effect of high temperatures can be excluded, because portedbyfairlygoodbootstrappingvalues.However,thelower
thehyperthermophilicPKfromThermotogashowedtheclassic valuesofsomebasalnodesareprobablyduetotheinfluenceof
allosteric response. The different regulatory behaviors in hy- several factors: phylogenetic distance, regulation, physiology,
perthermophilicarchaeaandThermotogamightbeduetothe evolutionary pressure, and temperature adaptation. The PK
differencesinglycolyticpathways,e.g.inarchaeaallEMpath- bacteria I cluster includes the majority of the bacterial PKs,
ways are modified, whereas in Thermotoga the classic EM andforthoseenzymes,whichwerefunctionallycharacterized,
pathway is operative. A comparative analysis of e.g. adenine AMPhasbeenshowntobeapositiveallostericeffector.Almost
nucleotidepoolsinhyperthermophilicarchaeaandThermotoga all archaeal PK sequences available form a separate cluster
mightgiveananswertothedifferentresponsetoAMP. supporting the monophyletic origin of the archaeal domain.
Sequence Alignment—PKs from the hyperthermophilic or- Interestingly, the PK from the hyperthermophilic bacterium
ganismsT.maritima,A.pernix,andP.aerophilumshowahigh T. maritima clusters within the archaeal sequences. This
degreeofsimilaritytocharacterizedandputativePKs(37–65% might reflect lateral gene transfer of the pyk gene from an
similarity)ofeukaryaandbacteria.Thus,allPKsconstitutea hyperthermophilic archaeon into the T. maritima genome, a
homologous family: besides minor deviations at variable posi- phenomenonthathasbeensuggestedtooccurinT.maritima
tions,allthehyperthermophilicPKscontainthePKconsensus at high frequency (41). PKs from the archaeal cluster did not
pattern [LIVAC]-X-[LIVM](2)-[SAPCV]-K-[LIV]-E-[NKRST]- showheterotropicregulationorwereregulatedbyAMP(Ther-
X-[DEQHS]-[GSTA]-[LIVM](40).Analignmentofthesehyper- motogaandThermoplasma).ThePKsequenceofthearchaeon
thermophilic PKs with selected homologous proteins from all A. fulgidus strain 7324 is not known. N-terminal amino acid
three domains is given in Fig. 6. Available PK crystal struc- sequences indicate high identity to PKs from the archaea
tures,fromcatandrabbitmuscle,yeast,E.coli,andL.mexi- Pyrococcus and Thermococcus. The absence of a pyk homo-
log in the genome of the closely related strain, A. fulgidus
cana revealed that each subunit is composed of four domains
VC16,mightbeexplainedbyalossofthisgene;alternatively,
(N, A, B, and C) (eukarya) or three domains (A, B, and C)
A.fulgidus7324mighthavetakenupitspykgenevialateral
(bacteria).ThedomainN,locatedattheNterminus,isashort
(cid:3)-helicalstretchpresentineukaryoticsequencesbutabsentin genetransferfromThermococcales.TheputativePKfromthe
Halobacterium NRC I, an outgroup with low bootstrapping
the bacterial and archaeal homologs. The domain A, which
support, was omitted. All eukaryotic PK sequences form a
includesthecatalyticsite(residues14–83and176–348ofthe
P. aerophilum PK, Fig. 6), constitutes a classic ((cid:3)(cid:1)) barrel cluster.MostofthemareallostericallyactivatedbyF-1,6-BPor
structure.ThedomainB(residues84–175)isa(cid:1)-sheet8capping F-2,6-BP (PKs from protistas). However, the absence of allo-
steric activation has been reported for a few eukaryal isoen-
the catalytic domain. The domain C (residues 349–461), lo-
cated at the C terminus, is an open twisted (cid:3),(cid:1) structure, zymes. The second bacterial PK cluster comprises PKs from
Gram-positives with low guanine-cytosine content and (cid:5)-pro-
containing the FBP binding site of the yeast enzyme. The
teobacteria species. Enzymes from this group show allosteric
highestdegreeofhomologyisfoundinthecatalyticdomainA.
responsetoeitherFBPortoAMP.Thetwoseparatebacterial
Inthisdomain,anumberofresidueshavebeenidentifiedtobe
PK clusters might have evolved by a gene duplication in the
important for catalysis, according to the yeast PK structure
early bacterial evolution. Alternatively, lateral gene transfers
(26).TheseresiduesareconservedinallselectedPKsequences,
from eukaryotes to some Gram-positive and proteobacteria
including the PKs of this study. In contrast to the domain A,
might be postulated. The latter hypothesis could explain the
domain B and in particular C showed significantly lower se-
closeclusteringofthesecondbacterialgroupwiththeeukarya,
quence homology. As deduced from the structure of the yeast
aswellastheallostericregulationbyFBPofisoenzymes1from
PK,atleasteightresidues(Ser-402,Thr-403,Ser-404,Thr-407,
E.coliandS.thyphimurium,apropertyfoundonlyineukaryal
Trp-452,Arg-459,Gly-475,andHis-491oftheyeastPK)have
PKs.
beenidentifiedinthedomainCtocontacttheallostericeffector
FBP. However, these residues are not conserved among the Acknowledgments—WethankDr.J.Gro¨tzinger(Kiel)forhelpinCD
FBP-regulated PKs. The presence of a conserved glutamate spectroscopicmeasurements,Dr.R.Schmid(Osnabru¨ck)forN-termi-
nal amino acid sequencing, and H. Preidel (Kiel) for mass culturing
(Glu-432)hasbeenattributedtothenon-allostericpropertyof
A.fulgidus7324andT.maritima.
the mammalian M1 isoenzyme (26). In the PKs of hyperther-
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