AMP-forming acetyl-CoA synthetases in Archaea show unexpected

14 Pages·2014·9.38 MB·English

Archaea2, 95–107
© 2006 Heron Publishing—Victoria, Canada
AMP-forming acetyl-CoA synthetases in Archaea show unexpected
diversity in substrate utilization
1 1,2
CHERYLINGRAM-SMITH and KERRY S. SMITH
1 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634-0318, USA
2 Corresponding author ([email protected])
Received May 15, 2006; accepted August 14, 2006; published online...
Summary Adenosine monophosphate (AMP)-forming acetate + ATP +CoA↔acetyl-CoA+ AMP +PP (1)
i
acetyl-CoAsynthetase(ACS;acetate:CoAligase(AMP-form-
ing),EC6.2.1.1)isakeyenzymeforconversionofacetateto Basedonisotopicexchange,labelingexperimentsanddetec-
acetyl-CoA,anessentialintermediateatthejunctionofanabo- tion of an enzyme-bound acetyl-AMP, a mechanism (Equa-
licandcatabolicpathways.Phylogeneticanalysisofputative tions 2a and 2b) in which the reaction proceeds through an
shortandmediumchainacyl-CoAsynthetasesequencesindi- acetyl-AMP intermediate has been proposed (Berg 1956a,
catesthattheACSsformadistinctcladefromotheracyl-CoA Berg 1956b, Webster 1963,AnkeandSpector1975):
synthetases. Within this clade, the archaeal ACSs are not
monophyleticandfallintothreegroupscomposedofbothbac-
E + acetate + ATP↔Eacetyl-AMP+PP (2a)
terialandarchaealsequences.Kineticanalysisoftwoarchaeal i
enzymes, an ACS from Methanothermobacter thermauto-
trophicus (designated as MT-ACS1) and an ACS from Eacetyl-AMP+HSCoA↔E +acetyl-CoA+ AMP(2b)
Archaeoglobus fulgidus (designated as AF-ACS2), revealed
thattheseenzymeshaveverydifferentproperties.MT-ACS1 Thefirststepofthereaction,whichrequiresacetateandATP,
hasnearly11-foldhigheraffinityand14-foldhighercatalytic butnotCoA,involvesformationoftheacetyl-AMPintermedi-
efficiencywithacetatethanwithpropionate,apropertyshared ateandreleaseofpyrophosphate(PP).Inthesecondstep,the
i
bymostACSs.However,AF-ACS2hasonly2.3-foldhigher acetylgroupistransferredtothesulfhydrylgroupofCoAand
affinityandcatalyticefficiencywithacetatethanwithpropio- AMPisreleased.Aninorganicpyrophosphatasedrawsthere-
nate.Thisenzymehasanaffinityforpropionatethatisalmost actioninthisforwarddirectionbyremovingPP,apotentin-
i
identical to that of MT-ACS1 for acetate and nearly tenfold hibitor of ACS.
higherthantheaffinityofMT-ACS1forpropionate.Further- The ACS is a member of the acyl-adenylate forming en-
more,MT-ACS1islimitedtoacetateandpropionateasacyl zyme superfamily in which all members undergo a similar
substrates,whereasAF-ACS2canalsoutilizelongerstraight two-step reaction mechanism with an enzyme-bound
andbranchedchainacylsubstrates.Phylogeneticanalysis,se- acyl-adenylateintermediateformedinthefirststepofthereac-
quencealignmentandstructuralmodelingsuggestamolecular tion.Althoughmembersofthissuperfamilyallcatalyzemech-
basis for the altered substrate preference and expanded sub- anistically similar reactions, they share little identity and
strate range of AF-ACS2 versus MT-ACS1. similarityinaminoacidsequencewiththeexceptionofafew
signaturemotifsandconservedcoresequencemotifs(Babbitt
Keywords:acetate,Archaeoglobusfulgidus,Methanothermo- et al. 1992, Kleinkauf and Von Dohren 1996, Chang et al.
bacterthermautotrophicus. 1997,Marahieletal.1997).Structuresforseveralmembersof
thisfamilyhavebeendetermined(Contietal.1996,Contiet
al. 1997, May et al. 2002), but provide little information re-
garding the active site and catalytic mechanismof ACS,be-
Introduction
cause they catalyze unrelated reactions in which the
Acetyl-CoAplaysacentralroleincarbonmetabolisminthe intermediates serve different functions and share too little
Bacteria, Archaea and Eukarya as an essential intermediate homology to allow structural modeling of ACS.
at the junction of various anabolic and catabolic pathways. The structures of the Salmonella enterica ACS and Sac-
Adenosinemonophosphate(AMP)-formingacetyl-CoAsyn- charomycescerevisiaeACS1nowprovidedirectinsightinto
thetase (ACS; acetate:CoA ligase (AMP-forming), EC the catalytic mechanism of ACS. The S. cerevisiae enzyme
6.2.1.1) is widespread in all three domains of life and is the wascrystallizedinthepresenceofATP(JoglandTong2004)
predominant enzyme for activation of acetate to acetyl-CoA andtheS.entericaenzyme(Gulicketal.2003)wascrystal-
(Equation 1). lized in the presence of CoA and adenosine-5′-propyl-
96 INGRAM-SMITH AND SMITH
phosphate, which mimics the acyl-adenylate intermediate tension penalty of 0.05. Aligned sequences were analyzed
(Grayson and Westkaemper 1988, Horswill and Escalante- withtheMEGAprogram(Kumaretal.1994)usinganeighbor
Semerena2002).Thesestructuresdemonstratetheenzymein joiningalgorithmwithagammadistanceestimation(γ =2).
two different conformations. The structure of the yeast en- Thephylogenywasconstructedbasedonpairwisedistancees-
zymeisthoughttorepresenttheconformationoftheenzyme timates of the expected number of amino acid replacements
inthefirststepofthereaction,whichinvolvesacetateandATP per site (0.2 in the scale bar). One thousand bootstrap repli-
butnotCoA.Inthisstructure,thesmallerC-terminaldomain cateswereperformedandvaluesof80%orhigherareshown.
isinanopenpositionawayfromtheactivesite.Thestructure Sequencesfrom onlyonestrainorspeciesofcloselyrelated
ofthebacterialenzymeisthoughttorepresenttheconforma- bacteriawereincludedintheanalysisforbrevityandreadabil-
tion for the secondstepof the reaction, in which the acetyl- ity.
adenylateintermediatereactswithCoA.Inthisstructure,the Sequencesimilarityandidentityweredeterminedusingthe
C-terminaldomainhasrotated140°towardtheN-terminaldo- BLAST2 pairwise alignment program (http://www.nc-
main, thus rearranging the active site upon CoA binding for bi.nlm.nih.gov/blast/bl2seq/wblast2.cgi). ConSurf (Armon
catalysis of the second step of the reaction. etal.2001,Glaseretal.2003,Landauetal.2005)(http://con-
MostACSshavealimitedsubstraterange,showingastrong surf.tau.ac.il) was used to determine evolutionary conserved
preferenceforacetateastheacylsubstrate,althoughpropio- residues that are likely to be important for protein structure
nate can serve as a less efficient substrate. However, the and function.
PyrobaculumaerophilumACS(PA-ACS)hasbeenshownto
utilizebutyrateandisobutyrateinadditiontoacetateandpro- Cloning and sequencing theacsgenes
pionate (Brasen et al. 2005). Furthermore, PA-ACS is
The genes encoding MT-ACS1 and MT-ACS2 from
octameric, unlike other ACSs which have been shown to be M. thermautotrophicus strains ΔH, Z245, and FTF, and
monomeric, dimeric or trimeric (Brasen et al. 2005). These
AF-ACS2fromA.fulgiduswerePCRamplifiedfromgenomic
findings call into question whether ACSs are more diverse
DNA using the following primer pairs: MT-ACS1,
than previously expected. 5′-ATGTCAAAGGATACCTCAGTTCTCC-3′ and 5′-CAT-
Wereportherethebiochemicalandkineticcharacterization CAAATATGAAGGGAGGGTATGG-3′; MT-ACS2, 5′-AT-
oftwoACSsfromthearchaeaMethanothermobactertherm- GAGAGGACAGCTTGATGCTCTG-3′ and 5′-CTGATTC-
autotrophicus(MT-ACS1)andArchaeoglobusfulgidus(AF- TCCCATCGGCAAATGG-3′;AF-ACS2,5′-ATGGCAGAC-
ACS2). MT-ACS1 is a typical ACS in that acetate is the CCGATGGAAGCTATG-3′ and 5′-CCACTTTGAAGCCAT-
stronglypreferredsubstrateoverpropionateanditcannotuti- ACTACCACC-3′. The PCR products were purified from
lize larger substrates such as butyrate. Through modeling of
agarosegelusingtheSpinPrepGelDNAKit(Novagen,Madi-
MT-ACS1 on the S. enterica and yeast ACS structures,
son,WI).ThePCRproductswereclonedintothepETBlue-1
Ingram-Smithetal.(2006)identifiedfourresiduesthatcom-
expressionvector(Novagen).Thesequencesoftheclonedacs
priseatleastpartoftheacetatebindingpocketofMT-ACS1
geneswere confirmed by Li-Cor bidirectional sequencingat
andhaveshownthatalterationsoftheseresiduescangreatly
the Nucleic Acid Facility at Clemson University.
influenceacylsubstrate range and preference.
AsobservedfortheP.aerophilumenzyme,AF-ACS2isun-
Heterologous enzyme production in Escherichia coli
usualinthatitshowsonlyaweakpreferenceforacetateversus
The enzymes MT-ACS1, MT-ACS2, and AF-ACS2 were
propionateandcanalsoutilizebutyrateandisobutyrate.The
heterologously produced in E. coli RosettaBlue(DE3). Cul-
presenceofthefouracetatepocketresiduesinbothAF-ACS2
tures were grown at 37 °C in LB medium containing 50 µg
andPA-ACS2suggeststhatadditionalresiduesplayanimpor-
ml–1ampicillinand34µgml–1chloramphenicoltoA =0.6.
tant role in determining substrate range and preference. The 600
Heterologousproteinproductionwasinducedbytheaddition
possiblemolecularbasisforthebroadsubstratespecificityof
of0.5mMIPTG.Cellsweregrownovernightat22–25°Cand
thesetwoenzymesrelativetoMT-ACS1andothercharacter-
harvested.
izedACSsis discussed.
Enzyme purification
Materials and methods A similar purification scheme was used for both MT-ACS1
andAF-ACS2.Cellssuspendedinice-coldbufferA(25mM
Sequence and phylogenetic analysis
Tris (pH 7.5)) were disrupted by two passages through a
Putative ACS amino acid sequences were identified in Frenchpressurecellat138MPaandthecelllysatewasclari-
BLASTPandTBLASTNsearches(Altschuletal.1990,1997) fiedbyultracentrifugation.Thesupernatantwasappliedtoa
of thefinishedgenomesequencesattheNationalCenterfor Q-sepharose fast-flow anion exchange column (GE Health-
BiotechnologyInformation(NCBI)usingtheM.thermauto- care,Piscataway,NJ)thatwasdevelopedwithalineargradient
trophicus Ζ245 MT-ACS1 deduced amino acid sequence as from0to1MKClinbufferA.Fractionscontainingactiveen-
thequery.Acetyl-CoAsynthetasesequenceswerealignedby zyme were pooled and diluted with 0.5volumesof buffer B
ClustalX(Thompsonetal.1997) usingaGonnetPAM250 (25mMTris(pH7.0))containing2Mammoniumsulfateand
weightmatrixwithagapopeningpenaltyof10.0andagapex- appliedtoaphenylsepharosefast-flowhydrophobicinterac-
ARCHAEAVOLUME 2, 2006
SUBSTRATE DIVERSITY OF ACETYL-COA SYNTHETASES 97
tioncolumn(GEHealthcare)thatwasdevelopedwithagradi- sion to fit the data to the Michaelis-Menten equation using
ent from 0.7 to 0 M ammonium sulfate in buffer B. The Kaleidagraph(SynergySoftware).TheenzymesfollowedMi-
purifiedenzymesweredialyzedagainstbufferBandconcen- chaelis-Menten kinetics for all substrates with the exception
tratedto>1mgml–1.Theenzymeswerepurifiedtoapparent that inhibition was observed above 0.5 mM HSCoA for
homogeneity as judged by sodium dodecyl sulfate poly- MT-ACS1, in which case the kinetic parameters for acetate
acrylamidegelelectrophoresis(SDS-PAGE)(Laemmli1970). andATPwereperformedinthepresenceof0.5mMHSCoAin
Aliquotsofthepurifiedproteinwerestoredat–20°C.Protein the reaction mixture.
concentrations were determined by the Bradford method
(Bradford 1976) with bovine serum albumin as the standard. Modeling the MT-ACS1 and AF-ACS2 structures
The M. thermautotrophicus MT-ACS1 and the A. fulgidus
Molecular mass determination AF-ACS2 structures were modeled on the S. enterica ACS
structure(PDBID:1PG4)usingDSModeler(AccelrysInc.,
Thenativemolecularmassofeachenzymewasdeterminedby
San Diego, CA) and the default parameters. The structures
gelfiltrationchromatographyonaSuperose12column(GE
werevisualizedwithDSVisualizer(Accelrys)andDSViewer
Healthcare) calibrated with chymotrypsinogen (25 kDa),
Pro5.0(Accelrys).Themodelswerevisuallycomparedwith
ovalbumin (43 kDa),albumin (67 kDa),aldolase(158 kDa),
the S. enterica ACS structure to ensure there were no major
catalase (232 kDa), ferritin (440 kDa) and blue dextran
structural anomalies.
(2000 kDa). Protein samples (0.2 ml) were loaded onto the
columnpre-equilibratedwith50mMTris(pH7.5)containing
Genbankaccession numbers
150mMKClandthecolumnwasdevelopedataflowrateof
0.5mlmin–1.Thesubunitmolecularmassofeachenzymewas MethanothermobacterthermautotrophicusΔHACS1(MTH-
217-MTH216), NP_275360 and NP_275359; and M. therm-
determinedbySDS-PAGEandwasinagreementwiththepre-
autotrophicusΔHACS2(MTH1603-MTH1604),NP_276715
dicted size based on the deduced amino acid sequence.
andNP_276716.M.thermautotrophicusZ245ACS1,DQ274-
062; and M. thermautotrophicus Z245 ACS2, DQ355203.
Enzymatic assays for ACS activity
M.thermautotrophicusFTFACS1,DQ355204;andM.therm-
Enzymaticactivitywasdeterminedbymonitoringacetyl-CoA autotrophicusFTFACS2,DQ355205;andA.fulgidusACS2,
formationfromacetate,ATPandCoAbythehydroxamatere-
NP_069202.
action(LipmannandTuttle1945,Roseetal.1954),inwhich
activated acyl groups are converted to an acyl-hydroxamate
andsubsequentlytoaferrichydroxamatecomplexthatcanbe Results
detectedspectrophotometricallyat540nm.Reactionmixtures
Presence of two ACS open reading frames (ORFs) in
contained 100 mM Tris (pH 7.5), 600 mM hydroxylamine-
M.thermautotrophicus
HCl(pH7.0)and2mMglutathione(reducedform),withvar-
iedconcentrationsofacylsubstrate,HSCoA,andMgCl -ATP. Analysis of the genome sequence of M. thermautotrophicus
2
Astandardreactiontemperatureof65°Cwasused,asthiswas ΔH revealed the presence of two putative ACSs, designated
determined to be the optimal temperature for both enzymes. hereasMTΔH-ACS1andMTΔH-ACS2.TheM.thermauto-
Reactionswereterminatedbytheadditionoftwovolumesof trophicusΔHgenomesequenceannotationindicatesthegene
stopsolution(1NHCl,5%trichloroaceticacid,1.25%FeCl). encodingMTΔH-ACS1isinterruptedbyastopcodonanda
2
Acetyl-CoA formation wasquantified by comparison with a frame shift, resulting in two adjacent ORFs (MTH217-
standard curve prepared using known concentrations of MTH216, gi:2621263 and 2621262) that together have
acetyl-CoA in the reaction mixture. Reaction times for each homologytofulllengthACS.TheDNAregionfromthestart
enzymewereempiricallydeterminedsuchthattherateofthe ATGofMTH217tothestopcodonofMTH216wasamplified
reactionremainedlinearandwaswithintheacetyl-CoAstan- and cloned into the pETBlue-1 expression vector. A soluble
dard curve. truncated protein of about 63 kDa was heterologously pro-
Fordeterminationofapparentkineticparameters,thecon- duced in E. coli but did not exhibit ACS activity (data not
centration of one substrate (acyl substrate, HSCoA or shown). This size is consistent with the position of the stop
equimolar MgATP) was varied and the other two substrates codon indicated in the published genome sequence. The se-
were held constant at saturating concentrations as follows: quenceoftheclonedM.thermautotrophicusΔHACS1gene
MT-ACS1, 40 mM acetate, 20 mM MgATP, 0.5 mM CoA; (determined concurrently with the overexpression studies)
AF-ACS2,50mMacetate,20mMMgATP,1mMCoA.Con- confirmed the stopcodonfor MTH217 is authentic.
centrationsforthevariedsubstrategenerallyrangedfrom0.2 The genes encoding the ACS1 homologs from M. therm-
to5–10timestheK value.Fordeterminationofapparentki- autotrophicus strains Z245 and FTF were also cloned for
m
neticparametersformetals,theATPconcentrationwasheldat heterologous expression in E. coli. The three MT-ACS1
20mMforbothenzymesandthemetalconcentrationswere homologs share 98.5% amino acid sequence identity, with
varied from 0.1 to 10mM. onlyninepositionsthatarenotidenticalamongallthree(data
Theapparentsteady-statekineticparametersk andk /K not shown). Sequence analysis of the genes encoding the
cat cat m
andtheirstandarderrorsweredeterminedbynonlinearregres- MT-ACS1homologsfromstrainsZ245andFTFrevealedthe
ARCHAEAONLINE at http://archaea.ws
98 INGRAM-SMITH AND SMITH
presenceofaGlucodonattheequivalentpositiontothestop TheS.entericaACSandS.cerevisiaeACS1sequences,rep-
codonintheM.thermautotrophicusΔHMT-ACS1.Alteration resenting the only two ACSs whose structures have been
ofthestopcodoninthegeneencodingM.thermautotrophicus solved (Gulick et al. 2003, Jogl and Tong 2004), reside in
ΔHMT-ACS1toaGlucodonresultedinproductionofafull Groups I and II. Among the putative archaeal ACSs, the se-
length,butinsoluble,proteinthatwasnotcharacterized(data quences of the Haloarcula marismortui ACS1 and P. aero-
not shown). philum ACS (PA-ACS), for which enzymatic activities have
The M. thermautotrophicus ΔH genome sequence annota- beenproven(BrasenandSchonheit2005,Brasenetal.2005),
tionforthegeneencodingMT-ACS2indicatesthatthisgeneis reside in Groups VII and VIII, respectively (Figure 1). The
alsointerruptedbyastopcodon,asforMT-ACS1,resultingin MethanosaetaconciliiACSsequenceinGroupVIIisidentical
twoadjacentORFs(MTH1603-MTH1604)thattogetherhave tothatoftheMethanothrixsoehngeniiACS,whichhasbeen
homologytofulllengthACS.TheDNAregionfromthestart purified and characterized (Jetten et al. 1989, Eggen et al.
ATGofMTH1603tothestopcodonofMTH1604wasampli- 1991).
fiedandclonedintothepETBlue-1expressionvector,aswere Toobtainamorethoroughrepresentationofthecharacteris-
thegenesencodingtheMT-ACS2homologsfromM.therm- tics of archaeal enzymes across the ACS phylogeny, the
autotrophicus strains Z245 and FTF. The three MT-ACS2 M.thermautotrophicusMT-ACS1andA.fulgidusAF-ACS2,
homologsshare97%identityindeducedaminoacidsequence, whosesequencesresideingroupsVIIandVIIIofthephylog-
with only 12–14 amino acid differences between any two eny(Figure1),werebiochemicallyandkineticallycharacter-
(datanotshown).SequenceanalysisoftheclonedM.therm- ized.
autotrophicus ΔH MT-ACS2 gene indicated an error in the
M. thermautotrophicus ΔH genome sequence. An additional Biochemical analysis of MT-ACS1 and AF-ACS2
nucleotideispresentthatshiftsthereadingframesuchthata
The enzymes MT-ACS1 and AF-ACS2 were heterologously
full length protein of 73kDais encoded.
producedinE.coliassoluble,activeproteinsandpurified.The
Heterologous expression of the M. thermautotrophicus
calculatedmassesfortheMT-ACS1andAF-ACS2monomers
Z245andFTFMT-ACS1genesinE.coliresultedinsoluble,
are 71,556 Da and 77,587 Da, respectively. The molecular
activeproteinsoftheexpectedsizeforafulllengthACS.The
massesofthenativeenzymesweredeterminedbygelfiltration
M.thermautotrophicusZ245 MT-ACS1(henceforth referred
chromatographytobe144.8kDaand221.2kDa,respectively,
toasMT-ACS1)waspurifiedtoelectrophoretichomogeneity
suggestingMT-ACS1isadimerandAF-ACS2isatrimer.The
andsubjectedtobiochemicalandkineticcharacterization.Ex-
temperatureoptimumwasdeterminedtobeabout65–70°C
pression of each of the three MT-ACS2 genes in E. coli re-
foreachenzyme(Figure2).Lessthan50%activitywasob-
sultedinproductionofa73kDaprotein(datanotshown),in
servedbelow45°Cforbothenzymes.At80°C,MT-ACS1re-
agreement with the size predicted for the full length ACS.
tained only 35% activity, whereas AF-ACS2 retained 44%
However, all three MT-ACS2 proteins were insoluble and
activity at 90 °C and still had 20% activity at 100 °C.
were not characterized.
Kinetic analysis of MT-ACS1 and AF-ACS2
Phylogenetic analysis of ACS
TheacylsubstraterangeforMT-ACS1waslimitedtoacetate
Phylogenetic analysis of putative short and medium chain
and propionate; the enzyme was unable to utilize butyrate.
acyl-CoA synthetase sequences revealed several distinct
clades, one of which contains all of the proven ACSs. Pro- However,AF-ACS2wasabletoutilizeacetate,propionateand
pionyl-CoA synthetase (Horswill and Escalante-Semerena butyrate.Thisenzymealsohadstrong,butunsaturable,activ-
2002), Sa (Fujino et al. 2001b) and MACS1 (Fujino et al. ity with isobutyrate and weak, but unsaturable, activity with
2001)acyl-CoAsynthetasesthatshowapreferenceforpropio- valerate, but could not utilize larger acyl substrates or other
nate, isobutyrate and octanoate, respectively, reside in other branchedchainacylsubstrates.Thekineticparametersdeter-
cladesoutsideoftheACSclade.WithintheACSclade,shown mined for each enzyme are shown in Table 1.
inFigure1,thesequencesformeightmajorgroups.MostACS Thereareanumberofnoteworthypointstobemadefrom
sequencesgroup according to domain. Groups II and III are theresultsinTable1.Thetwoenzymeshavesimilaraffinity
composedsolelyofeukaryoticsequences,withtheexception (Km) for acetate but very different affinities for propionate.
of a single bacterial sequence in each. There are three large TheaffinityofAF-ACS2forpropionateisalmostidenticalto
groups (I, IV and V) composed exclusively of bacterial se- thatofMT-ACS1foracetateandnearlytenfoldhigherthanthe
quencesandseveralsmallbacterialclusters.Thearchaealse- affinityofMT-ACS1forpropionate.Whereasthedifferencein
quences fall into three groups (VI, VII and VIII), each of affinity between the two substrates is over tenfold for
which also contains one or more bacterial sequences (Fig- MT-ACS1, AF-ACS2 has only a 2.3-fold higher affinity for
ure 1). acetatethanpropionate.MT-ACS1hasastrongpreferencefor
Figure1(facingandfollowingpage).PhylogenyofACSsequences.Aphylogenyofputativeshortandmediumchainacyl-CoAsynthetasesfrom
thefinishedgenomesequencesavailableatNCBIwasconstructedusingtheneighborjoiningalgorithmofMEGA(Kumaretal.1994).Onlythe
majorcladecontainingtheprovenACSsisshownhere.Formostgenera,sequencesfromonlyonespecieswereusedinconstructingthephylog-
eny for brevity and readability. Eukaryotic sequences are indicated in black, bacterial sequences in red andarchaealsequences in blue.
ARCHAEAVOLUME 2, 2006
SUBSTRATE DIVERSITY OF ACETYL-COA SYNTHETASES 99
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100 INGRAM-SMITH AND SMITH
ARCHAEAVOLUME 2, 2006
SUBSTRATE DIVERSITY OF ACETYL-COA SYNTHETASES 101
Figure2.TemperatureoptimaforMT1-ACSandAF-ACS2.Enzyme Figure3.DivalentmetalspecificityforMT1-ACSandAF-ACS2.En-
reactionswereperformedattheindicatedtemperaturesintriplicate. zymereactionswereperformedintriplicateat65°Cinthepresenceof
Activitiesarereportedasapercentageofthemaximumactivitydeter- 20mMmetal(asthechloridesalt)+20mMATP.Activitiesarere-
mined for each enzyme. Symbols: (cid:2) = MT1-ACS; and (cid:3) = AF- portedasapercentageofthemaximumactivitydeterminedforeach
ACS2. enzyme with Mg2+as the metal substrate.
acetateasthesubstrateasshownbythe14-foldhighercata- similar for both enzymes. Both enzymes demonstrated a
lyticefficiency(k /K )withacetateversuspropionate.How- strongpreferenceforATPversusCTP,GTP,TTP,UTP,ITPor
cat m
ever, AF-ACS2 has only a 2.3-fold higher preference for ADP,forwhichlessthan5%activitywasobserved(datanot
acetateoverpropionate.Finally,AF-ACS2wasabletoutilize shown).
butyrateassubstrate,althoughtheK was78-foldhigherthan The metal specificity was tested for each enzyme using a
m
thatforacetateand34-foldhigherthanthatforpropionate,and standardmetalconcentrationof20mMand20mMATP.Both
theturnoverratewas21-foldreducedwithbutyratecompared MT-ACS1andAF-ACS2showedstrongpreferenceforMg2+
with acetate or propionate. andMn2+asthedivalentmetal(Figure3)andCo2+alsogave
TheK valuesforCoAforbothenzymesshowedlessthan high activity. Strong activity was observed with Ca2+ for
m
twofolddifference(Table1),andtheK valuesforATPwere AF-ACS2 but not for MT-ACS1. Moderate activity was ob-
m
served for both enzymes with Ni2+, whereas Cu2+ and Zn2+
workedpoorlyforbothenzymes.Thekineticparametersde-
termined for those metals that gave the highest activity are
Table 1. Kinetic parameters for MT-ACS1 and AF-ACS2. showninTable2.ForMT-ACS1,thehighestaffinityandturn-
overratewereobservedwithMg2+.Althoughthehighestturn-
Substrate Enzyme K k k /K
m cat cat m over rate was observed with Mg2+, Mn2+ and Ca2+ gave the
(mM) (s–1) (s–1mM–1)
highest catalytic efficiencies for AF-ACS2.
Acetate MT-ACS11 3.5 ± 0.1 65.4 ± 0.3 18.6 ± 0.5
AF-ACS2 1.7 ± 0.06 35.9 ± 0.58 21.2 ± 0.4
Propionate MT-ACS11 36.5 ± 1.9 46.3 ± 0.7 1.3 ± 0.04
Discussion
AF-ACS2 3.9 ± 0.02 35.7 ± 0.17 9.1 ± 0.06
Butyrate MT-ACS11 — — — Although our kinetic and biochemical characterization of
AF-ACS2 133.0 ± 14.6 1.68 ± 0.07 0.013 ± 0.0009
MT-ACS1andAF-ACS2expandsourknowledgeoftheprop-
Valerate MT-ACS1 — — —
ertiesofACSs,thereisstillapaucityofinformation onthis
AF-ACS2 Unsaturable
importantclassofenzymes.Withtheadventofwholegenome
Isobutyrate MT-ACS1 — — —
AF-ACS2 Unsaturable sequencing, gene functions are usually assigned based on
ATP MT-ACS11 3.3 ± 0.2 66.6 ± 0.9 20.2 ± 0.9 homologywithothersequencesinthesequencedatabases.In
AF-ACS2 2.9 ± 0.01 38.1 ± 0.12 13.2 ± 0.09 manycases,aparticularenzymaticfunctionmaybeassigned
CoA MT-ACS1 0.19 ± 0.003 81.6 ± 0.7 423.7 ± 4.7 basedonhomologytojustasinglesequencewhosefunction
AF-ACS2 0.30 ± 0.003 44.3 ± 0.2 144.9 ± 1.2 hasbeenproven.Acetyl-CoAsynthetaseiswidespreadinall
1KineticparametersforMT-ACS1weredeterminedinthepresence three domains, and most putative ACSs have been assigned
of 0.5mMCoA. this function through homology. Of the 193 ACS sequences
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102 INGRAM-SMITH AND SMITH
Table 2. Kinetic parameters for divalent metals for MTI-ACS and During growth on glucose, these halophiles excreted acetate
AF-ACS2. into the media and exhibited ADP-forming acetyl-CoA
synthetaseactivity(ADP-ACS;acetyl-CoA+ADP+P ↔ac-
Enzyme Metal K k k /K i
m cat cat m etate+ATP+CoA)butnotACSactivity.Uponentryintosta-
(mM) (s–1) (s–1mM–1)
tionary phase, ACS activity was induced and the excreted
MT1-ACS1 Mg2+ 0.38 ± 0.02 56.5 ± 1.0 147.5 ± 4.5 acetatewasconsumed.Thus,ADP-ACSwasdeterminedtobe
Mn2+ 0.61 ± 0.02 44.6 ± 0.5 72.8 ± 2.3 responsible for acetate and ATP production from excess
Co2+ 0.99 ± 0.03 39.4 ± 0.3 40.0 ± 1.1
acetyl-CoAduringgrowthonglucosebutACSwasresponsi-
AF-ACS2 Mg2+ 1.08 ± 0.05 23.4 ± 0.3 21.8 ± 0.7
ble for activation of acetate for use as a carbon and energy
Mn2+ 0.70 ± 0.02 20.6 ± 0.23 29.7 ± 0.76
Ca2+ 0.64 ± 0.006 18.2 ± 0.05 28.3 ± 0.27 source.
Co2+ 1.50 ± 0.02 19.0 ± 0.13 12.2 ± 0.09 AlthoughtheH.marismortui,M.soehngenii,andM.ther-
mophila CALS-1 ACSs and the M. thermautotrophicus
1KineticparametersforMT-ACS1weredeterminedinthepresence
MT-ACS1allshowastrongpreferenceforacetate,thecharac-
of 0.5mMCoA.
terizedACSsfromA.fulgidusandP.aerophilumhaveanex-
pandedsubstraterange.Whatphysiologicalpurposecouldthis
broadsubstraterangeserve?OnepossibilityisthatA.fulgidus
showninthephylogenyinFigure1,onlyahandfulhavebeen andP.aerophilumcanutilizeamorediversearrayofcarbonor
biochemicallycharacterized.TheS.entericaACSandS.cere- energy sources than other archaea. Both P. aerophilum and
visiaeACS1,whosestructureshavebeensolved(Gulicketal. A.fulgiduscanutilizecomplexorganicssuchasyeastextract,
2003,JoglandTong2004),arequitedistantfromthearchaeal meat extract, tryptone, and peptone as growth substrates
ACSs, for which there is no structure. (Stetter 1988, Volkl et al. 1993), whereas the others cannot.
Although genes predicted to encode for ACS are wide- ThepresenceofanACSwithanexpandedsubstraterangemay
spread in the Archaea, only a few archaeal ACSs have been provideameansforutilizationofothershortchainfattyacids
biochemicallycharacterized.Acetyl-CoAsynthetaseactivity eitherscavengedfromtheenvironmentorfromthebreakdown
was first detected in archaea in Methanothermobacter ofcomplexorganicswithouttheneedforadditionalenzymes.
marburgensis (formerly Methanobacterium thermoautotro- The findings of the phylogenetic analysis presented here
phicum Marburg) (Oberlies et al. 1980), a thermophilic (Figure1)contrastwiththoseofBrasenandSchonheit(2005)
chemolithoautotrophic methanoarchaeon that can utilize with respect to the archaeal ACSs. In their analysis, the
H /CO as the sole carbon and energy source (Zeikus and archaeal sequencesformed one distinct clade, leading to the
2 2
Wolfe 1972). When M. marburgensis (closely related to conclusionthatthearchaealsequencesformaseparatebranch
M.thermautotrophicus)wasgrownonH /CO inthepresence withintheprokaryoticsequencesandhaveamonophyleticori-
2 2
of acetate, 10% of cellular carbon was derived from acetate gin(BrasenandSchonheit2005).Inouranalysis,thearchaeal
with the remainder derived from CO (Fuchs et al. 1978). sequencesformthreegroups,eachofwhichalsocontainsbac-
2
Oberliesetal.(1980)subsequentlydemonstratedACSactivity terialsequences.Thismaybearesultofthelargernumberof
inM.marburgensiscellsgrownwithlimitingH /CO andpro- sequences from each domain used in this analysis (193 se-
2 2
posedthatACSallowsassimilationofacetateasacellularcar- quences composed of 107 bacterial, 33 archaeal, and 53
bon source in order to spare limited supplies of CO. eukaryoticsequencesversus51totalsequencesbyBrasenand
2
The first archaeal ACSs purified and characterized were Schonheit(2005))anddifferencesinmethodology.However,
those from M. soehngenii and Methanothrix thermophila aseparatephylogeneticanalysisusingtheminimumevolution
CALS-1 (now Methanosaeta concilii and Methanosaeta algorithmoftheMEGApackage(Kumaretal.1994)showed
thermophila CALS-1) (Jetten et al. 1989, Teh and Zinder a similar result (data not shown).
1992).Acetyl-CoAsynthetaseisthefirstenzymeintheactiva- Overall, the ACSs show strong sequence conservation re-
tion of acetate to acetyl-CoA for methanogenesis in the gardless of domain, as indicated by pairwise amino acid se-
obligatelyacetoclasticMethanosaeta(Jettenetal.1989,Teh quencecomparisonsthatshowMT-ACS1andAF-ACS2share
andZinder1992,AllenandZinder1996).Infact,therecently 38–49% identity and 57–67% similarity with S. cerevisiae
completedgenomesequencesofMethanosaetaspeciesreveal ACS1andS.entericaACS.However,thesubunitcomposition
that M. thermophilaP hasfour genesthat encodeACSand oftheACSsisquitediverse,withmonomeric(S.entericaand
T
M.conciliihasfive(K.S.SmithandC.Ingram-Smith,unpub- H.marismortuiACSs(BrasenandSchonheit2005,Gulicket
lisheddata).AllofthemethanoarchaeawithACShaveatleast al. 2003), dimeric (MT-ACS1, M. concilii, and Bradyr-
two acs genes, with the exception of Methanococcus hizobium japonicum ACSs (Jetten et al. 1989, Preston et al.
maripaludis(Figure 1). 1990,Leeetal.2001)),trimeric(AF-ACS2andS.cerevisiae
Anumberofhalophilicarchaeaareabletoutilizeacetateas ACS1 (Jogl and Tong 2004)), and octameric enzymes
a carbon and energy source. Brasen and Schonheit (Brasen (PA-ACS(Brasenet al. 2005)) thus far characterized.
and Schonheit 2001, Brasen and Schonheit 2004) demon- CharacterizedenzymesfromgroupsI,II,III,VI,andVII,
strated that Halococcus saccharolyticus, Haloferax volcanii, whichincludethebacterialACSsfromS.enterica(Gulicket
HalorubrumsaccharovorumandH.marismortuigrownonac- al. 2003) and B. japonicum (Preston et al. 1990, Lee et al.
etate as a carbon and energy source exhibited ACS activity. 2001),theeukaryoticACS1andACS2enzymesfromhuman
ARCHAEAVOLUME 2, 2006
SUBSTRATE DIVERSITY OF ACETYL-COA SYNTHETASES 103
(Luongetal.2000,Fujinoetal.2001a)andS.cerevisiae(van ACS, MT-ACS1, and H. marismortui ACS sequences and a
den Berg et al. 1996), and archaeal ACSs from M. concilii partial alignment is shown in Figure 4. ConSurf analysis
(M. soehngenii) (Jetten et al. 1989, Eggen et al. 1991), (Armonetal.2001,Glaseretal.2003,Landauetal.2005)of
H.marismortui(BrasenandSchonheit2005),andM.therm- thealignmentwasperformedtohelpdelineateaminoacidres-
autotrophicum(MT-ACS1),allshowastrongpreferencefor idues that might contribute to the broad substrate range of
acetateastheacylsubstrate,withpropionatebeingtheonlyal- AF-ACS2andPA-ACSandsuggestwhethermembersofthis
ternative acyl substrate. AF-ACS2 and the P. aerophilum subcladewithingroupVIIIrepresentsasubclassofACSswith
PA-ACS(Brasenetal.2005),bothfromgroupVIII,havean expanded substrate range. ConSurf calculates evolutionary
expanded substrate range that includes butyrate and the conservationscoresforeachresiduewithinaproteinsequence
branchedchainisobutyrateaswellasacetateandpropionate. basedonproteinstructure,multiplesequencealignment,evo-
AF-ACS2showsonlyaweak(twofold)preferenceforacetate lutionary distance between sequences, and evolutionary tree
overpropionate,unlikemostACSsthatgenerallyhavea10-to topology. The evolutionary conservation scores are then
20-foldpreferenceforacetate,asdeterminedbythehighercat- mappedontotheproteinstructuretodefineprobableregions
alytic efficiency (k /K ). of structural and functional importance.
cat m
AlthoughafullkineticcharacterizationofPA-ACSwasnot Using the S. enterica ACS structure as the query for
reported,theK valueforacetatewasdeterminedtobe3µM, ConSurfanalysis,theevolutionaryconservationscoreforeach
m
over 500-fold lower than that observed for AF-ACS2. Al- positionalongwiththealternativeresiduesfromthefiftymost
thoughthismayindicatethattheseenzymeshaveverydiffer- closely related sequences were determined. Salmonella
entkineticproperties,itmayalsobeduetodifferencesinthe entericaACSresiduesdeterminedtobethemosthighlycon-
enzymeassayusedinthetwostudies.TheK valueforacetate servedbyConSurfanalysisareshadedinthepartialalignment
m
observed for PA-ACS is at least 20- to several hundredfold (Figure4),alongwithresiduesintheothersequencesthatare
lowerthanthatobservedforanyotherACS.TheK valuefor identicaloramongthealternativeresiduesforthatposition.In
m
acetateforAF-ACS2iswellinlinewiththevaluesdetermined the complete alignment, eleven residues with high ConSurf
usingthehydroxamateassaywithotherarchaealACSsinclud- scores(allofwhichareshowninthepartialalignmentinFig-
ingMT-ACS1(Jettenetal.1989,Prestonetal.1990,Tehand ure4)areconservedinthethreeACSsequencesrepresenting
Zinder 1992). It is not known whether these differences are enzymes with “traditional” characteristics, but differ in
meaningfulwithregardtowhethertheseenzymesmayrepre- AF-ACS2 and PA-ACS. In addition, the four Sulfolobus se-
sentasubgroupofACSsthatshowonlyweakpreferencefor quences that reside in the same subclade in group VIII as
acetate and expanded substrate range. AF-ACS2andPA-ACSofthephylogenyalsodifferatthese
ThesefindingsleadonetoquestionwhetherAF-ACS2and same positions.
PA-ACSareanomalieswithintheACSsorwhethertheyrepre- MT-ACS1 and AF-ACS2 have been modeled on the
sent a subset of enzymes with different properties from the S. enterica ACS structure to determine whether the acetate
“traditional” ACSs that have a strong preference for acetate bindingpocketsshowanymajordifferencesthatcouldbeat-
and a narrow substrate range. Four residues (Ile312, Thr313, tributedtothedifferencesinsubstratepreferenceandsubstrate
Val388,andTrp416)havebeenshowntoformtheacetatebinding rangeofthesetwoenzymes.ThemodelsdepictedinFigure5
pocketofMT-ACS1andhavebeenshowntobeimportantin show residues within 10 Å of the propyl group of the
acylsubstrateselection(Ingram-Smithetal.2006).Alteration adenosine-5′-propylphosphateligand.IntheS.entericastruc-
of any of these four residues influences substrate affinity or ture, the adenosine-5′-propylphosphate mimics the acetyl-
substraterange,orboth,aswellascatalysis(Ingram-Smithet adenylateintermediateandthepropylgroupapproximatesthe
al.2006).Forexample,alterationofTrp416toGly,theresidue positionofacetateintheactivesite(Gulicketal.2003).Over-
foundinshortandmediumchainacyl-CoAsynthetasesother all,theacetatebindingsitesofthetwomodeledenzymesare
thanacetyl-andpropionyl-CoAsynthetases,expandsthesub- verysimilar(Figure5).However,therearekeydifferencesin
straterangeofMT-ACS1suchthattheenzymecanutilizesub- thepositioningofcertainresiduesthatareconservedinboth
strates ranging from acetate to octanoate (including some enzymesandinresiduesthatwereidentifiedbytheConSurf
branchedchainacylsubstrates)andchangesthesubstratepref- analysis(Figure4)tobeconservedinthetraditionalACSsbut
erencefromacetatetovalerate.Inpropionyl-CoAsynthetase, nottheAF-ACS2/PA-ACSsubcladeofgroupVIII(Figure1).
Val388isreplacedbyAla.AVal388AlaMT-ACS1variant has The propyl group is in a similar position in both the
higher affinity for propionate than acetate and a slightly MT-ACS1 and AF-ACS2 models, and three of the acetate
greaterpreferenceforpropionateaswell.AmongtheACSse- pocket residues occupy similar positions as well. However,
quencesinFigure1,includingbothAF-ACS2andPA-ACS, whereas Ile312 of MT-ACS points away from the propyl-
Thr313,Val388,andTrp416arecompletelyconservedandIle312is phosphate group (Figure 5A), Ile329 of AF-ACS2 points in-
highly conserved,with Val asthe only other amino acid ob- wards(Figure5B).Thismayincreasethehydrophobicityof
served at the equivalent position. theacetatepocketandcouldaccountinpartforthehigheraf-
Within group VIII in the ACS phylogeny in Figure 1, a finity of AF-ACS2 for acyl substrates than observed for
subcladeconsistingofAF-ACS2andPA-ACSaswellasfour MT-ACS1. Among residues identical to both enzymes, the
Sulfolobussequencesisstrongly supportedbybootstrapping othermajordifferenceobservedintheacetatepocketregionis
(84%). These sequences were aligned with the S. enterica that Leu424 of MT-ACS1 is positioned quite differently from
ARCHAEAONLINE at http://archaea.ws
104 INGRAM-SMITH AND SMITH
Figure 4. Alignment and
ConSurfanalysis of ACS se-
quences. TheM.thermauto-
trophicusMT-ACS1 and
A.fulgidusAF-ACS2 amino
acid sequences were aligned
with theS.entericaACS se-
quence (SE-ACS) and the ACS
sequences fromP.aerophilum
(PA-ACS),Sulfolobustokodaii
(ST-ACS1 and ACS2),
Sulfolobussolfataricus(SS-
ACS), andSulfolobusacido-
caldarius(SA-ACS) using
ClustalX(Thompson et al.
1997). A partial alignment is
shown here. Residues of the
S.entericaACS found to have
high evolutionary conservation
scores byConSurfanalysis
(Armonet al. 2001,Glaseret
al. 2003, Landau et al. 2005)
(http://consurf.tau.ac.il) are
shaded, as are residues in the
other ACS sequences that are
identical or among the alterna-
tive residues listed for each of
these highly conserved posi-
tions. Asterisks indicate those
positions that are identical in
all nine sequences. The acetate
binding pocket residues are
boldfaced and numbered above
the aligned sequences accord-
ing to their position within
MT-ACS1. Those residues at
positions with highConSurf
scores that differ in AF-ACS2,
PA-ACS, and theSulfolobus
sequences from the three ACS
sequences representing en-
zymes with “traditional” char-
acteristics are indicated in red.
the equivalent residue Leu441 of AF-ACS2. The overall in- Ile402,His403,Ser429,Ser430,andHis444ofAF-ACS2)areclus-
creasedhydrophobicityoftheacylsubstratebindingpocketof teredtoonesideofthepocketforbothenzymes(Figure5)and
AF-ACS2maypositivelyinfluencesubstrateaffinity,butitis mayinfluencethepositioningofGlu390,whichpointsslightly
likely that the combined effects of multiple residues are re- inwardtowardthepocketinMT-ACS1,andtheequivalentres-
quiredtodeterminewhethertheenzymehasanarroworbroad idueGlu407ofAF-ACS2thatpointsslightlyawayfromtheac-
substrate range. etatepocket.Withdrawalofthenegativechargeofthisresidue
OftheelevenresiduesidentifiedbyConSurfanalysistobe fromtheacetatepocketinAF-ACS2maypositivelyinfluence
highly conserved in traditional ACSs but not the AF- substrate binding.
ACS2/PA-ACSsubclade(Figure4),sixofthesepositionsare Thesixthresidue,representedbyGln417ofMT-ACS1and
in close proximity to Val388 and Trp416 of MT-ACS1 Met434ofAF-ACS2,ismoreintimatelypositionedneartheac-
(Figure5A),thetwoacetatepocketresiduesthatwereshown etatepocket(Figure5).Glutamine417pointsawayfromtheac-
to have the greatest influence on substrate range and prefer- etate pocket and the propylphosphate group, whereas Met434
ence (Ingram-Smith et al. 2006). Five of these residues bendsintowardthepropylphosphate.Met434mayplayarolein
(Leu385, Gly386, Ile412, Asp413, and Pro427 of MT-ACS1 and expandingthesubstraterangeofAF-ACS2byextendingthe
ARCHAEAVOLUME 2, 2006