Role of 4-Hydroxybutyrate-CoA Synthetase in the CO2 Fixation Cycle in Thermoacidophilic Archaea

12 Pages·2013·1.56 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.288,NO.6,pp.4012–4022,February8,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Role of 4-Hydroxybutyrate-CoA Synthetase in the CO
2
Fixation Cycle in Thermoacidophilic Archaea*
Receivedforpublication,August24,2012,andinrevisedform,October26,2012 Published,JBCPapersinPress,December20,2012,DOI10.1074/jbc.M112.413195
AaronS.Hawkins‡1,YejunHan‡,RobertK.Bennett‡,MichaelW.W.Adams§,andRobertM.Kelly‡2
Fromthe‡DepartmentofChemicalandBiomolecularEngineering,NorthCarolinaStateUniversity,Raleigh,NorthCarolina
27695-7905andthe§DepartmentofBiochemistryandMolecularBiology,UniversityofGeorgia,Athens,Georgia30602
Background: Thermoacidophilic Sulfolobales contain a novel CO fixation pathway; all enzymes but one have been
2
accountedforinMetallosphaerasedula.
Results:EnzymesencodedinMsed_0394andMsed_0406eachexhibit4-hydroxybutyrate-CoAsynthetaseactivity,consistent
withtranscriptomicevidence.
Conclusion:Msed_0406islikelythephysiologicallyrelevantenzymeinthecycle.
Significance:AllenzymesarenowaccountedforintheCO fixationcycleofM.sedula.
2
Metallosphaera sedula is an extremely thermoacidophilic Carbondioxideischemicallystableandunreactiveandmust
archaeon that grows heterotrophically on peptides and chemo- be reduced to enable its incorporation into biological mole-
lithoautotrophically on hydrogen, sulfur, or reduced metals as cules. Autotrophic microorganisms are able to utilize carbon
energy sources. During autotrophic growth, carbon dioxide is dioxideastheirsolecarbonsource,andavarietyofpathways
incorporatedintocellularcarbonviathe3-hydroxypropionate/4- are known to activate and incorporate it into biomolecules
hydroxybutyratecycle(3HP/4HB).Todate,allofthestepsinthe essentialforgrowthandreplication.Recently,carbondioxide
pathway have been connected to enzymes encoded in specific fixation pathways have received interest for biotechnological
genes,exceptfortheoneresponsibleforligationofcoenzymeA applications, since this could provide biological routes for de
novogenerationoffuelsandsmallorganicmolecules(1).
(CoA)to4HB.Althoughseveralcandidatesforthisstephavebeen
There are currently at least six natural pathways for the
identifiedthroughbioinformaticanalysisoftheM.sedulagenome,
incorporationofinorganiccarbondioxideintocellularcarbon
nonehavebeenshowntocatalyzethisbiotransformation.Inthis
(2,3).Themostrecentlydiscoveredofthesearefoundexclu-
report,transcriptomicanalysisofcellsgrownunderstrictH -CO
2 2 sivelyinextremelythermophilicarchaeaasfollows:the3-hy-
autotrophywasconsistentwiththeinvolvementofMsed_0406and
droxypropionate/4-hydroxybutyrate(3HP3/4HB)carbonfixa-
Msed_0394.Recombinantversionsoftheseenzymescatalyzedthe
tion cycle, which operates in members of the crenarchaeal
ligationofCoAto4HB,withsimilaraffinitiesfor4HB(K valuesof
m orderSulfolobales(2,4–6),andthedicarboxylate/4-hydroxy-
1.9and1.5mMforMsed_0406andMsed_0394,respectively)but butyrate(DC/4HB)cycle,whichisusedbyanaerobicmembers
with different rates (1.69 and 0.22 (cid:1)mol (cid:2) min(cid:3)1 (cid:2) mg(cid:3)1 for
oftheordersThermoprotealesandDesulfurococcales(4,7).In
Msed_0406andMsed_0394,respectively).NeitherMsed_0406nor
bothcycles,twocarbondioxidemoleculesareaddedtoacetyl-
Msed_0394haveclosehomologsinotherSulfolobales,although
CoA(C2)toproducesuccinyl-CoA(C4),whichissubsequently
lowsequencesimilarityisnotunusualforacyl-adenylate-forming rearrangedtoacetoacetyl-CoAandcleavedintotwomolecules
enzymes.Thecapacityofthesetwoenzymestouse4HBasasub- of acetyl-CoA. These pathways differ primarily in regard to
stratemayhavearisenfromsimplemodificationstoacyl-adeny- theirtolerancetooxygenandtheco-factorsusedforreducing
late-formingenzymes.Forexample,asingleaminoacidsubstitu- equivalents as follows: NAD(P)H for the 3HP/4HB cycle and
tion (W424G) in the active site of the acetate/propionate ferredoxin/NAD(P)H for the DC/4HB cycle (3, 8). The two
synthetase (Msed_1353), an enzyme that is highly conserved archaealpathwaysalsodifferinhowtheylinktheCO fixation
2
amongtheSulfolobales,changeditssubstratespecificitytoinclude cycletocentralmetabolism.IntheDC/4HBpathway,pyruvate
4HB. The identification of the 4-HB CoA synthetase now com- is synthesized directly from acetyl-CoA using pyruvate syn-
pletesthesetofenzymescomprisingthe3HP/4HBcycle. thase.Inthe3HP/4HBpathway,anotherhalf-turnisrequired
tomakesuccinyl-CoA,whichisthenoxidizedviasuccinateto
pyruvate(2,9,10).
There are 13 enzymes proposed to catalyze the 16 reac-
*Thisworkwassupported,inwholeorinpart,byNationalInstitutesofHealth tionsinthe3HP/4HBpathway.Thefirstthreeenzymescon-
Grant R01GM90209. This work was also supported by Department of
vert acetyl-CoA (C2) to 3HP (C3) via an ATP-dependent
EnergyAdvancedResearchProjectsAgency-EnergyGrantDE-AR0000081
andDefenseThreatReductionAgencyGrantHDTRA1-09-1-0030. carboxylation step. Next, 3HP is converted and reduced to
1SupportedbyGraduateAssistanceinAreasonNationalNeedMolecular propionyl-CoA,carboxylatedasecondtime,andrearranged
Biotechnology Fellowship P200A070582-09 from the United States
DepartmentofEducation.
2To whom correspondence should be addressed: Dept. of Chemical and
BiomolecularEngineering,NorthCarolinaStateUniversity,911Partners 3Theabbreviationsusedare:3HP,3-hydroxypropionate;4HB,4-hydroxybu-
Way,EB-1,Box7905,Raleigh,NC27695-7905.Tel.:[phone];Fax:919- tyrate;DC,dicarboxylate;ACL,autotrophiccarbon-limited;HTR,hetero-
515-3465;E-mail:[email protected]. trophic;ACS,acetyl-CoAsynthetase;ACR,autotrophiccarbon-rich.
This is an Open Access article under the CC BY license.
4012 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER6•FEBRUARY8,2013
CO -H AutotrophyinMetallosphaerasedula
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TABLE1
Enzymesinthe3HP/4HBcycleinM.sedula
Thefollowingabbreviationsareused:NCE,nativecellextract;NP,nativepurifiedenzyme;R,recombinantprotein.
Cycleref.no. ORF Enzyme Ref.
E1(cid:2) Msed_0147 Acetyl-CoA/propionyl-CoAcarboxylase NCE(11,41)
E1(cid:1) Msed_0148
E1(cid:3) Msed_1375
E2 Msed_0709 Malonyl-CoA/succinyl-CoAreductase R(42)
E3 Msed_1993 Malonatesemialdehydereductase R(42)
E4 Msed_1456 3-Hydroxypropionate:CoAligase NP(5)
E5 Msed_2001 3-Hydroxypropionyl-CoAdehydratase NP,R(43)
E6 Msed_1426 Acryloyl-CoAreductase NP(43)
E7 Msed_0639 Methylmalonyl-CoAepimerase R(13)
E8(cid:2) Msed_0638 Methylmalonyl-CoAmutase R(13)
E8(cid:1) Msed_2055
E9 Msed_1424 Succinatesemialdehydereductase NP,R(42)
E10 Msed_0394 4-Hydroxybutyrate-CoAsynthetase R(thiswork)
Msed_0406
E11 Msed_1321 4-Hydroxybutyryl-CoAdehydratase NCE(4)
E12 Msed_0399 Crotonyl-CoAhydratase/(S)-3-hydroxybutyryl-CoAdehydrogenase R(9)
E13 Msed_0656 Acetoacetyl-CoA(cid:1)-ketothiolase NCE(4)
to make succinyl-CoA (C4). Succinyl-CoA is reduced to Msed_1291hadnoactivityonanyofthepreviouslymentioned
4HB,whichisconvertedtotwomoleculesofacetyl-CoAin organic acids. Thus, although cycle function has been con-
the final reactions of the cycle. Flux analysis and labeling firmedbymetabolicfluxanalysis,andalthough4HB-CoAsyn-
studieshaveconfirmedtheoperationofthispathwayinMet- thetase activity has been measured in cell extracts of
allosphaerasedula(4,10). autotrophically grown M. sedula, the enzyme responsible for
Alloftheenzymesthatcomprisethefirstportionofthecycle ligationofCoAto4HBremainsunclear.
uptotheformationof4HBhavebeenidentifiedandcharacter- Toidentifythemissinglinkinthe3HP/4HBcycle,newmeth-
izedbiochemicallyintheirnativeorrecombinantform,mostly odsforsemi-continuouscultivationofM.sedulainagas-inten-
from the extremely thermoacidophilic archaeon M. sedula sivefermentationsystemweredevelopedtoteaseoutdifferen-
(T (cid:1) 70°C, pH 2.0) (see Table 1) (4, 5, 11–13). The enzymes tialtranscriptionalresponseofautotrophy-relatedgenes.Strict
involvedintheconversionof4HBtotwomoleculesofacetyl- carbon dioxide limitation was used to drive increased opera-
CoA have not been characterized to the same extent (Fig. 1). tionalefficiencyoftheCO fixationenzymes,whichhypothet-
2
Activities corresponding to 4-hydroxybutyryl-CoA dehydra- ically would increase transcriptional levels of genes encoding
taseandacetoacetyl-CoA(cid:1)-ketothiolasehavebeendetectedin key enzymes to maximize carbon incorporation. Using these
cellextracts(4,14),althoughneitherenzymehasbeenpurified conditionsfortranscriptionalanalysis,amuchclearerpicture
initsnativeformorrecombinantlyproduced.Identificationof emergedconcerningtheglobalregulatorychangesinM.sedula
candidatesforbothoftheseenzymeshasbeenmadebasedon asitscellularmetabolismswitchesfromautotrophytohetero-
genomeannotationandtranscriptomicanalysisofautotrophic trophy. This strategy produced new leads for the genes and
growthcomparedwithheterotrophy(8,9).Althoughneitherof correspondingenzymesresponsibleforthe4HB-CoAligation
the candidate genes for these enzymes has so far been con- step.Theenzymeswererecombinantlyproducedandshownto
firmedbiochemically,theiridentityisnotindisputebecauseof catalyzetheligationofCoAto4HB.
stronghomologytoknownversionsinlessthermophilicorgan-
EXPERIMENTALPROCEDURES
isms. The corresponding gene products in M. sedula are
Msed_1321forthe4HB-CoAdehydrataseandMsed_0656for Growth of M. sedula in a Gas-intensive Bioreactor—M.
theacetoacetyl-CoA(cid:1)-ketothiolase. sedula(DSMZ5348)wasgrownaerobicallyat70°Cinashak-
The identity of the crotonyl-CoA hydratase and the (S)-3- ingoilbath(90rpm)underautotrophicorheterotrophiccon-
hydroxybutyryl-CoA dehydrogenase was recently confirmed ditionsonDSMZmedium88atpH2.Heterotrophicallygrown
whenitwasdiscoveredthatbothreactionswerecatalyzedbya cellsweresupplementedwith0.1%tryptone.Cellgrowthwas
singlebifunctionalfusionprotein(9).Inthesamework,Ramos- scaledupfrom300mlinsealed1-literbottles(seeRef.8)to2
Vera et al. (9) tested three different candidates for the 4HB- litersinastirredbench-topglassfermentor(Applikon),alsoon
CoAsynthetase,buttheyallfailedtoshowactivityon4HB.In DSMZmedium88,pH2,at70°C,andagitatedat250rpm.Two
fact,theprimarycandidatesuggestedbytheautotrophictran- separately regulated gas feeds were used such that flow rates
scriptomeanalysis(Msed_1422)showednoenzymaticactivity wereheldconstantforallconditionsat1ml/minforthehydro-
on short chain linear unsubstituted or hydroxy acids, specifi- gen/CO gasmixtures(compositionvaried)and100ml/minfor
2
cally acetate, propionate, 3HP, 3-hydroxybutyrate, 4HB, and air (composition: 78% N , 21% O , 0.03% CO ). For the
2 2 2
crotonate. Two other candidates were selected, based on autotrophic carbon-rich (ACR) condition, the gas feed con-
homology to 4HB-CoA synthetase from Thermoproteus neu- tained H (80%) and CO (20%); for the autotrophic carbon-
2 2
trophilus (Tneu_0420) and 3HP-CoA synthetase from M. limited(ACL)condition,thefeedwaschangedtoH (80%)and
2
sedula: Msed_1353 and Msed_1291 were recombinantly pro- N (20%);fortheheterotrophiccondition(HTR),themedium
2
ducedandtestedforligaseactivity.Msed_1353wasactiveon wassupplementedwith0.1%tryptone,andthegasfeedcompo-
propionate and acetate, but not on 4HB. Furthermore, sitionwasN (80%)andCO (20%).Tandemfermentorswere
2 2
FEBRUARY8,2013•VOLUME288•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 4013
CO -H AutotrophyinMetallosphaerasedula
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FIGURE1.Enzymesandsubstratesinfinalreactionsof3HP/4HBcycleinM.sedula.Enzymesareasfollows:10,4-hydroxybutyrate-CoAsynthetase;11,
4-hydroxybutyryl-CoAdehydratase;12,crotonyl-CoAhydratase/(S)-3-hydroxybutyryl-CoAdehydrogenase;and13,acetoacetyl-CoA(cid:1)-ketothiolase.
FIGURE2.Bioreactorschematicforgas-intensivefermentationofM.sedula.Tandem2Lbioreactors,startedatthesametimewiththesameseedinoculum,
wereusedtogrowM.sedulainsideofachemicalfumehood.AsolenoidvalveontheH /CO tankprovidedpassive“fail-safe”operationbycuttingofftheflow
2 2
offlammablegasintheeventoffoodfailure.Gascompositionsforthethreedifferentconditionsareshownbottomright.ACR,autotrophiccarbon-rich;ACL,
autotrophiccarbon-limited;HTR,heterotrophic.
runsimultaneouslywiththesameinoculumtogeneratebiolog- photometricandoneusinghighperformanceliquidchroma-
ical repeats (Fig. 2). Cells were harvested at mid-exponential tography(HPLC).Adiscontinuousassaywasusedtomeasure
phasebyrapidcoolingwithdryiceandethanolandthencen- substrate-dependentdisappearanceofCoAat75°C.Thereac-
trifugedat6000(cid:2)gfor15minat4°C. tionmixture(600(cid:4)l)contained100mMMOPS/KOH,pH7.9,5
M. sedula Oligonucleotide Microarray Transcriptional Re- mMMgCl ,2.5mMATP,0.15mMCoA,andpurifiedenzyme.At
2
sponse Analysis—A spotted whole-genome oligonucleotide eachtimepoint,80(cid:4)lofreactionmixturewasaddedto80(cid:4)lof
microarray,basedon2256protein-codingopenreadingframes cold5,5(cid:4)-dithiobis-(2-nitrobenzoicacid).Atimepoint(0min)
(ORFs),wasused,asdescribedpreviously(15).TotalRNAwas wastakenbeforeheating.Thereactionmixturewasincubated
extracted and purified (RNeasy; Qiagen), reverse-transcribed for2minat75°C,followedbyadditionofsubstrate.Additional
(Superscript III; Invitrogen), re-purified, labeled with either timepointsweretakenat30,60,90,120,and180safteraddi-
Cy3orCy5dye(GEHealthcare),andhybridizedtothemicroar- tionofsubstrate.Absorbancewasmeasuredat412nmtodeter-
rayslides(Corning).SlideswerescannedonaGenePix4000B mine free CoA concentration, based on the concentration of
MicroarrayScanner(MolecularDevices,Sunnyvale,CA),and 2-nitro-5-thiobenzoate dianion ((cid:5) (cid:1) 14,150 M(cid:5)1 cm(cid:5)1) (1,
412
rawintensitieswerequantitatedusingGenePixProversion6.0. 14).Enzymeswerekineticallycharacterizedbyvaryingthecon-
Normalizationofdataandstatisticalanalysiswereperformed centration of the acyl-CoA substrate from 0.05 to 12 mM,
usingJMPGenomics5(SAS,Cary,NC).Ingeneral,significant although the other substrate concentrations were held con-
differentialtranscriptionwasdefinedtobearelativechangeat stant.Measurementsforspecificactivityweretakenundersat-
or above 2 (where a log value of (cid:3)1 equals a 2-fold change) urating substrate concentrations (10 mM). Formation of the
2
withsignificancevaluesatorabovetheBonferronicorrection; CoAesterwasalsoconfirmedusingHPLC(Waters).Thereac-
forthesedata,thiswas5.4(equivalenttoapvalueof4.0(cid:2)106). tionmixture(0.15ml)contained100mMpotassiumphosphate,
MicroarraydataareavailablethroughtheNCBIGeneExpres- pH 7.9, 10 mM MgCl , 2 mM ATP, 0.5 mM CoA, 10 mM sub-
2
sionOmnibus(GEO)underaccessionnumberGSE39944. strate,andpurifiedenzyme.Thereactionwasincubatedfor3
Enzyme Assays for 4-Hydroxybutyrate-CoA Synthetase— minat75°C,quenchedwith15(cid:4)lof1MHCl,filteredwitha
Twoassayswereusedtomeasureligaseactivity,onespectro- 10-kDaspincolumn(AmiconYM-10)toremovetheprotein,
4014 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER6•FEBRUARY8,2013
CO -H AutotrophyinMetallosphaerasedula
2 2
and loaded onto a reversed-phase C18 silica-based column KOH(pH7.9)andeitherstoredat4°Cormixedwithglycerol
(ShodexC18–4E,4.6(cid:2)250mm).Themobilephasewas50mM to20%andstoredat(cid:5)20°C.
sodiumphosphatebuffer,pH6.7,with2%methanol. Site-directed Mutagenesis of Msed_1353—Msed_1353 was
Heterologous Expression of M. sedula Genes in E.coli—M. mutated with the GENEART(cid:1) site-directed mutagenesis sys-
sedula genes encoding acyl-CoA synthetases were amplified tem (Invitrogen), using AccuPrimeTM Pfx polymerase.
from genomic DNA using primers synthesized by Integrated MutagenesisprimersweredesignedtochangeTrp424toglycine
DNA Technologies (Coralville, IA). Msed_0394 and Msed_ (primer 1, 5(cid:4)-CCCTTTGGTAGCACTTGGGGAATGACT-
0406 were ligated into pET46-Ek/LIC, although Msed_1353 GAAACTGG-3(cid:4);primer2,reversecomplementofprimer1).
wasligatedintopET21busingNdeIandXhoIrestrictionssites. Plasmids with Msed_1353-G424 were cloned into NovaBlue
All constructs were designed to express with an N-terminal GigaSinglesE.colicompetentcellsandselectedbygrowthon
His tag. Plasmids containing gene inserts were cloned into LB-agarsupplementedwithampicillin(100(cid:4)g/ml).Sequences
6
NovaBlue GigaSingles E.coli competent cells and selected by wereconfirmedbyEtonBiosciencesInc.(Durham,NC).
growthonLB-agarsupplementedwithampicillin(100(cid:4)g/ml). Structural Modeling of Acyl-CoA Synthetases—Three-di-
PlasmidDNAwasextractedusingaQIAprepspinminiprepkit. mensionalstructuralmodelsforM.sedulaacyl-CoAsyntheta-
ses were made using the iterative threading assembly refine-
SequenceswereconfirmedbyEtonBiosciences,Inc.(Durham,
ment (I-TASSER) on-line server (2, 3, 16). The server first
NC). For protein expression, the plasmids were transformed
generates three-dimensional atomic models from multiple
into Escherichia coli Rosetta 2 (DE3) cells and selected by
growthonLB-agar,supplementedwithampicillin(100(cid:4)g/ml) threading alignments and iterative structural assembly and
andchloramphenicol(50(cid:4)g/ml).Cellsharboringtherecombi- then infers function by structural matching to other known
nantplasmidwereinducedwithisopropyl1-thio-(cid:1)-D-galacto- proteins.AllstructuresweregeneratedusingtheProteinData-
baseentryforSalmonellaentericaACS(STM4275,1PG4)asa
pyranoside (final concentration 0.1 mM) atA 0.4–0.6 and
600 threading template for additional restraint specification.
culturedfor3hbeforeharvest.
Amino acid sequence alignments were generated using the
PurificationofRecombinantProteins—Cellswereharvested
by centrifugation at 6000 (cid:2) g for 15 min at 4°C. Cell yields UCSFChimerapackagebysuperpositionofI-TASSERthree-
dimensional structural models with the Protein Data Bank
ranged from 1.6 to 3.8 g of cells/liter of LB medium (wet
structureforS.entericaACS.
weight). Cell pellets were resuspended in lysis buffer (50 mM
Materials—Plasmidvectorsandstrainswereobtainedfrom
sodiumphosphate,100mMNaCl,0.1%NonidetP-40,pH8.0)
Novagen(SanDiego)andStratagene(LaJolla,CA).Chemicals,
containing DNase and lysozyme at final concentrations of 10
devices, and reagents were obtained from Fisher, ACROS
and 100 (cid:4)g/ml, respectively. Cells were lysed with a French
Organics (Geel, Belgium), Sigma, New England Biolabs (Ips-
press(twopassesat18,000p.s.i.),andthelysatewascentrifuged
wich, MA), Qiagen (Valencia, CA), Millipore (Billerica, MA),
at22,000(cid:2)gfor15minat4°Ctoremovedinsolublematerial.
and Invitrogen. Gases were purchased from Airgas National
Soluble,cell-freeextractwasheatedto65°Cfor20mintopre-
Welders (Charlotte, NC). Protein purification columns were
cipitatemesophilicproteins.Streptomycinsulfate(1%w/v)was
obtained from GE Healthcare. The Bradford assay reagent
addedtoprecipitatenucleicacids,followedbya1-hincubation
wasobtainedfromBio-Rad.Site-directedmutagenesiskitwas
at4°C.Afinalcentrifugationwasperformedat22,000(cid:2)gfor
obtainedfromInvitrogen.
15 min at 4°C to collect the soluble, heat-treated cell-free
extract,whichwassterile-filtered(0.22(cid:4)m)andpurifiedusing RESULTS
a 5 ml HisTrapTM nickel column (GE Healthcare). Proteins
M. sedula Autotrophic Growth Is Hydrogen-limited—To
wereboundtotheHisTrapTMcolumnusingbindingbuffer(50
exploretheoptimalgrowthconditionsforH -CO autotrophy
2 2
mMsodiumphosphate,500mMNaCl,20mMimidazole,pH7.4) inM.sedula,afermentationsystemwasdesignedtoallowcon-
andelutedusingelutionbuffer(50mMsodiumphosphate,500 trolled definition of the gas feed. Previous autotrophic work
mM NaCl, 300 mM imidazole, pH 7.4). SDS-PAGE was then withM.sedulawasdoneinbatchculturesinanorbitalshaking
performedontheimmobilizedmetalaffinitychromatography bath at 70°C (2, 4–6, 8). In that case, gas-fed cultures were
fractions to qualitatively determine the purity of the protein grown by replacing the air in a sealed volume with a gaseous
beforefurtherpurification.Chromatographyfractionscontain- mixtureofaknowncomposition.MasstransferofH ,CO ,and
2 2
ingtheproteinwereconcentrated,exchangedintophosphate O intotheculturemediumwaslimitedtodiffusionacrossthe
2
buffer (50 mM potassium phosphate, 150 mM NaCl, pH 7.0) vapor-liquid interface. Gas limitation presumably affected
using an Amicon YM10 (Millipore) centrifugal filter mem- thesecultures,andledtosuboptimalgrowth,asevidencedby
brane, and centrifuged at 4000 (cid:2) g and 4°C. To quantify the theslowdoublingtimethatresultedforM.sedulaunderthese
amountofprotein,aBradfordassaywasperformedonthecon- conditions(t (cid:1)11–13h).
d
centratedimmobilizedmetalaffinitychromatographyfractions TogrowM.sedulaautotrophicallywithmoreoptimaldeliv-
usingknownserialdilutionsofbovineserumalbumin(BSA)by eryofgaseoussubstratetotheliquidmedium,asemi-continu-
takingabsorbancereadingsat595nm.Proteinwasfurtherpuri- ousfermentationsystemwasdevelopedusinga3Lbioreactor.
fiedusingaSuperdex20010/300GL(GEHealthcare)gelfiltra- The system was modified to have two separate gas feeds that
tion column. The proteins were eluted from the gel filtration sparged directly into the media (sparging stone, 2-(cid:4)m pore
columnusingelutionbuffer(50mMpotassiumphosphate,150 size).Microbubblespargingstoneswereusedtopromotedis-
mMNaCl,pH7.0).Proteinsweredialyzedinto100mMMOPS- solutionofsparinglysolublegases,inparticularH .Thebiore-
2
FEBRUARY8,2013•VOLUME288•NUMBER6 JOURNALOFBIOLOGICALCHEMISTRY 4015
CO -H AutotrophyinMetallosphaerasedula
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TABLE2
EnhancedtranscriptionresponseforM.sedulaautotrophy
ACLversusACR ACLversusHTR ACRversusHTR AutoversusHetero(8)
No.ofgenesup-regulated(2-foldormore) 52 467 433 229
No.ofgenesdown-regulated(2-foldormore) 124 517 464 252
actorandconsoleweresituatedinsideamodifiedfumehood, tion proteins in both the cytosolic hydrogenase operon
withanairflowmonitoringsysteminplacetodetecthoodfail- (Msed_0921-0933) and the membrane-bound hydrogenase
ure.Tandemfermentorswereseededwiththesameinoculum operon(Msed_0947–0950)werebothhighlyup-regulatedon
andrunsimultaneouslytoprovideabiologicalrepeat. ACL-HTR,from3-to47-foldhigher.
GrowthofM.sedulainanaerobic,autotrophicfermentation New Candidates for 4-Hydroxybutyrate-CoA Synthetase
systemwasexpectedtobeH -andnotO -limited.Belowsat- Identified from Refined Transcriptomic Data—The refined
2 2
urating conditions, growth rates varied according to the transcriptomic data provided new insights into the putative
amountofH fedtotheculture.ForhighH supplyrates(i.e.30 candidates for 4-hydroxybutyrate-CoA synthetase (Fig. 3).
2 2
ml/min), the growth rates were comparable with the fastest Basedonbioinformaticanalysis,thereareninecandidategenes
growth rates previously observed under heterotrophy (t (cid:1) encoding acyl-CoA synthetases (not including Msed_1456,
d
4.8h);concomitantly,theculturereachedacelldensityof2(cid:2) whichwasconfirmedasa3HP-CoAsynthetase).Thehighup-
109 cells/ml, the highest observed under autotrophic condi- regulationofMsed_1422underautotrophy(13-foldincrease)
tions.AtanH supplyrateof15ml/min,thegrowthrateslowed thatwasobservedinthisworkisconsistentwithprevioustran-
2
(t (cid:1)6h),althoughthefinaldensitywascomparablewiththe scriptomic studies. On the basis of that initial study,
d
30ml/mincase(1.5(cid:2)109cells/ml).A30-foldreductioninH Msed_1422waschosenforrecombinantexpressionandtesting
2
flowrates(1ml/min)causedthegrowthratetodecreasebyhalf (2, 9, 9, 10). In the same study, recombinant forms of
(t (cid:1)9.7h)andthecellstoenterstationaryphaseat8(cid:2)108 Msed_1291 and Msed_1353 were also produced, which were
d
cells/ml. chosenbasedonhomologytoaconfirmed4HB-CoAsynthe-
AsimilartrendemergedinresponsetolimitinglevelsofCO . tasefromT.neutrophilus(Tneu_0420).Noneoftheseenzymes
2
WhenCO wassupplementedinthegasfeed(referredtohere showedactivityon4HB.Msed_1422andMsed_1291showed
2
as“rich”autotrophy),thegrowthratewasfasterthanobserved noactivityonacetate,propionate,3HP,3HB,4HB,orcroton-
forcellsgrownwithairastheonlysourceofCO (t (cid:1)6.8h ate,andMsed_1353hadactivityonlyonacetateandpropionate
2 d
versus 9.4 h, respectively). The growth rate for heterotrophi- butnot4HB.Thus,itappearsthatMsed_1353isapromiscuous
cally grown cells (t (cid:1) 6.7 h) was comparable with the rich acetate/propionatesynthetase,althoughthesubstratespecific-
d
autotrophycondition.Thissuggeststhat,undertherichautot- itiesofMsed_1422andMsed_1291remainunknown.
rophycondition,thecellswerenotlimitedbyanyoneparticular Amongtheotherpotentialcandidatesthatwereannotatedas
gaseoussubstrateandweredoublingatorneartheirmaximal acetate-CoAsynthetasesormediumchainfattyacid-CoAsyn-
rate.Thedecreaseingrowthrateforthecarbon-limitedautot- thetases(Fig.3),mostshowednotranscriptionalresponse,had
rophyarisesfromthelimitingamountsofCO availableinthe average or low levels of transcription, or were clearly down-
2
medium. regulatedunderautotrophy.Thenewtranscriptomicdatawere
OptimizedH -CO AutotrophyConditionsLeadtoEnhanced consistentwiththeexpressionoftwopreviouslyunexamined
2 2
TranscriptomicResponse—Theoptimizedautotrophicgrowth candidates, Msed_0406 and Msed_0394, that are annotated
conditionsenhancedtheglobaltranscriptionalresponsecom- asanacetyl-CoAsynthetase(ACS)andAMP-dependentsyn-
paredwithpreviouswork(4,7,8).Ofthe2293protein-coding thetase and ligase, respectively. Although Msed_0406 and
genesinthe2.2-kbM.sedulagenome,nearlyhalf(984genes) Msed_0394werebothconstitutivelytranscribed,withlessthan
exhibitedchangesintranscription(eitherup-ordown-regula- a2-foldchangeintranscriptionlevelsbetweentheconditions
tion)of2-foldorgreater,whencomparingheterotrophy(HTR) tested,bothofthemwereinthetop25%ofthetranscriptome.
withtheACLcondition(seeTable2).Thenumberofgenesthat This served as the basis to investigate these two genes by
were differentially transcribed was twice as high as observed recombinantexpressionandactivityassays,giventhatnoother
previously (3, 8, 8), which could be attributed to the refined promisingcandidatesforthisstephademerged.
conditions for autotrophic growth. Also, in the experiments KineticAnalysesofMsed_0394andMsed_0406—Recombi-
reportedhere,itshouldbementionedthattheimprovedsensi- nant forms of Msed_0394 and Msed_0406 were produced in
tivity of new equipment used for scanning microarray slides E.coli and purified to electrophoretic homogeneity. For both
improvedtheresolutionanddynamicresponse. enzymes,theproductionof4HB-CoAfrom4HBandCoAwas
Overall, the global transcriptional changes were extensive. confirmed using reversed-phase HPLC. Msed_0394 and
Transcripts for the characteristic enzymes of the 3HP/4HB Msed_0406wereactiveonarangeofsmallorganicacids(see
pathway were significantly up-regulated on ACL-HTR. For Table3forasummaryofkineticdata).Fig.4showstherelative
example,thegenesencoding(cid:2)-and(cid:1)-subunitsofacetyl-CoA/ specific activities on different substrates for Msed_0394 and
propionyl-CoAcarboxylase(Msed_0147–0148)wereup-regu- Msed_0406,alongwithreporteddatafor3HP-CoAsynthetase
lated18-and29-fold,respectively,whereasthe4-hydroxybu- (Msed_1456)forcomparison(4,5,10).Notethatthecalculated
tyryl-CoA dehydratase gene (Msed_1321) was up-regulated molecularmassforthesethreeenzymesvariesslightly,62kDa
27-fold.Hydrogenasesandhydrogenaseassemblyandmatura- for Msed_0394, 64 kDa for Msed_0406, and 74 kDa for
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FIGURE3.4-Hydroxybutyrate-CoAsynthetasecandidatesinM.sedula.NormalizedtranscriptionlevelsforM.sedulagenesannotatedassmallorganicacid
orfatty-acidligasesandsynthetases.Hightranscriptionlevelsareshowninredandlowtranscriptioningreen;thecorrespondingnumbersrepresentleast
squaremeanvaluesofnormalizedlog2-transformedtranscriptionlevelsrelativetotheoverallaveragetranscriptionlevelof0(black).Conditionsshownareas
follows:2010,Heterotrophic,Autotrophic,Mixotrophic;2012,ACL,ACR,HTR.LeastsquaremeanvaluesareshownhereforACLconditionforthesegenes,along
withthefoldchangeofgenesunderACLrelativetoHTRandtheirstatisticalsignificance.AllothermicroarraydatacanbefoundintheGEOdepositGSE39944.
Msed_1456;thesespecificactivitiesherearemeanttohighlight TABLE3
substratepreferencepatternsforeachenzyme. EnzymekineticdataforCoAsynthetasesfromM.sedula
ThespecificactivitiesforMsed_0394showlittledifferencein Enzyme Substrate K V k k /K
m max cat cat m
themaximumreactionrateundersaturatingsubstrateconcen- (cid:4)M (cid:4)mol s(cid:5)1 s(cid:5)1
trationsforthedifferentsubstrates.Thehighestreactionrate mmign(cid:5)(cid:5)11 M(cid:5)1
observedwas(cid:6)0.2(cid:4)molmin(cid:5)1mg(cid:5)1forpropionate,4HB,and Msed_0394 Acetate 680 0.13 0.14 200
butyrate.However,ifthesubstratespecificitiesaretakeninto Propionate 540 0.2 0.21 390
3HP 1880 0.07 0.08 40
account,adifferentpictureemerges.Acomparisonofthecat- 4HB 1540 0.22 0.24 160
alytic specificity constants (k /K ) for each substrate tested Butyrate 60 0.21 0.23 3700
cat m Valerate 120 0.2 0.22 2000
withMsed_0394(Table3)showsthatthehighestvalueisfor
Msed_0406 Acetate 2030 6.0 6.4 3200
butyrate(3700M(cid:5)1s(cid:5)1),followedbyvalerate(2000M(cid:5)1s(cid:5)1), Propionate 380 15.1 16.2 43000
propionate (390 M(cid:5)1 s(cid:5)1), acetate (200 M(cid:5)1 s(cid:5)1), and finally 3HP 810 2.4 2.6 3200
4HB (160 M(cid:5)1 s(cid:5)1). There is a clear preference for unsubsti- 4BHutByrate 2302000 17..79 18..84 92160000
tutedstraightchainorganicacidswithachainlengthoffouror Valerate 740 5.2 5.6 7500
five carbons. No activity was detected with the six-carbon Msed_1353-G424 4HB 1130 2.3 2.5 2180
hexanoicacid.
ThespecificactivitiesforMsed_0406undersaturatingsub- limitsthesizeofsubstratethatcanbeaccommodatedwithin
strateconcentrationsshowthehighestreactionratesforpro- theactivesite.Totesttheimportanceofthisresidueindeter-
pionate(15.1(cid:4)molmin(cid:5)1mg(cid:5)1).Thecatalyticspecificitycon- miningsubstratespecificity,Trp424inMsed_1353wasmutated
stantprofileforMsed_0406showsthatthisenzymeworksbest toaglycinetoproduceMsed_1353-G424.Thesinglesubstitu-
onpropionate(43,000M(cid:5)1s(cid:5)1)andthenbutyrate(26,000M(cid:5)1 tionmutant(W424G)waspredictedtocontainalargerinterior
s(cid:5)1),valerate(7500M(cid:5)1s(cid:5)1),acetate/3HP(3200M(cid:5)1s(cid:5)1),and binding pocket for the hydrophobic end of the substrate.
4HB(910M(cid:5)1s(cid:5)1).ThehighV valuesforacetate/propio- Accordingly, it showed a dramatic change in specificity (Fig.
max
nate,combinedwiththelowK valueforpropionate,suggest 5B). Activity for the mutant on acetate and propionate
m
that Msed_0406 is also a promiscuous acetate/propionate decreasedby60%,from8.9to3.6and8.8to3.5(cid:4)molmin(cid:5)1
ligase,althoughonethatalsoshowsactivityon4HB. mg(cid:5)1, respectively. However, Msed_1353-G424 also showed
Site-directed Mutagenesis of Msed_1353—Msed_1353, a activityonC4-C8substrates,including4HB(1.8(cid:4)molmin(cid:5)1
highlyconservedgeneamongtheSulfolobales,waspreviously mg(cid:5)1).
reportedtohaveactivityonlyonacetateandpropionate(4,5,5, Tocomparetheactivityofthesethreeenzymeson4HB,the
9,11,12,15).Initialeffortstoidentifytheunknown4HB-CoA Michaelis-MentencurvesareshowninFig.6.Fromthisfigure,
synthetaseinM.sedulainvolvedpurificationofnativeenzyme itisclearthatthereisalargedifferenceincatalyticrateforthe
activityandanalysisofmultipleSDS-polyacrylamidegelbands threeenzymes,andthisdifferenceholdsovertheentirerangeof
using mass spectrometry. Msed_1353 was detected in these substrate concentration, including when [S]/K (cid:7)(cid:7)1. There-
m
experiments, and based on the very large up-regulation of fore, although it is possible that both Msed_0394 and
Msed_1353underautotrophy,itwasrecombinantlyproduced Msed_0406 are catalytically active on 4HB in vivo, it is likely
toconfirmitsactivity.Ourresultsconfirmedpreviousreports; that Msed_0406 is more physiologically relevant in terms of
undersaturatingsubstrateconcentrations,Msed_1353hadthe catalyticperformance.Additionally,thesinglepointmutation
highestactivityonacetate(8.9(cid:4)molmin(cid:5)1mg(cid:5)1,100%)and ofMsed_1353toMsed_1353-Gly424producesanenzymethat
propionate (99%) but also on 3HP (8%) and butyrate (16%). is active on 4HB at even higher rates for all substrate
However,noactivitywasfoundon4HBorlongerorganicacid concentrations.
substrates(seeFig.5A).
DISCUSSION
Structural modeling of the binding pocket of Msed_1353
revealedaconservedtryptophanresidue,similartothatseenin Thesemi-continuousgas-intensivebioreactorsystemdevel-
acetate-CoAsynthetase(ACS)fromS.enterica(4,14,17).This oped here was successfully used to refine the transcriptional
tryptophanformsthebottomsurfaceofthebindingpocketand responseofautotrophy-relatedgenesinM.sedula.Thissystem
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FIGURE4.Specificactivityofacyl-CoAligasesintheM.sedulacarbonfixationpathwayonvarioussubstrates.Specificactivitiesofthenewcandidatesfor
4-hydroxybutyrate-CoA ligase on a variety of substrates compared with reported data for Msed_1456, a 3-hydroxypropionate-CoA ligase, as follows:
Msed_0394(A),Msed_0406(B),andMsed_1456(C).Msed_1456showed(cid:8)1%activityon3-hydroxybutyrate,butitwasnottestedon4-hydroxybutyrate.
Substrateabbreviations:ACE,acetate;PRO,propionate;3HP,3-hydroxypropionate;4HB,4-hydroxybutyrate;BUT,butyrate;VAL,valerate.
FIGURE5.SpecificactivityofnativeMsed_1353andMsed_1353-W424Gmutantonvarioussubstrates.ComparisonofactivityofMsed_1353(A)and
Msed_1353-G424(B)onavarietyofshortchainlinearorganicacids.Substrateabbreviations:ACE,acetate;PRO,propionate;3HP,3-hydroxypropionate;4HB,
4-hydroxybutyrate;BUT,butyrate;VAL,valerate;HEX,hexanoate;OCT,octanoate.
providedbetterdeliveryofgaseswithlowsolubilityandallowed limitsgrowth.Initsnaturalenvironment,thepicturemaybe
more precise regulation of gas composition than could be somewhatdifferent.Hydrogenmeasurementsfromthe(largely
achievedinserumbottles.At70°Cand1atm,thesolubilityof anoxic)acidichotspringsatYellowstoneindicatethatgaseous
oxygen and hydrogen are comparable (0.6 mM), although the hydrogenmaybequiteabundant,withconcentrationsranging
solubilityofcarbondioxideisabout20-foldhigher(12mM)(8, between10and300nM(8,19).Thesourceofthishydrogengas
9,18).Fortheseexperiments,thelowsolubilityofH wasoffset isprimarilygeochemical;althoughthemechanismisnotwell
2
bytheuseofmicrobubblersparingstones(2(cid:4)mporesize)to understood, it probably arises from subsurface interaction of
increasethegasphasesurfaceareaandincreasedeliveryofH waterwithFe[II](15,20).Formostsubsurfaceenvironments,
2
tothemedium. oxygenisprobablylimiting(21).However,M.sedulawasiso-
Stoichiometrically, at least four H molecules are required lated from aerobic (surface) samples of a hot water pond at
2
for every carbon atom fixed. Assuming that ATP generation PisciarelliSolfatara,Naples,Italy(22).Thus,bothhydrogenand
requires the oxidation of two hydrogen molecules, then each oxygenmaybeavailableinabundanceforautotrophicgrowth.
turnofthecyclerequires12moleculesofhydrogenforevery TheregulationofgrowthmodesinM.sedulainvolvesmas-
twomoleculesofcarbondioxide.Assuch,thelimitinggrowth sive transcriptional changes between heterotrophic and
factorforM.sedulainabioreactorislikelyacquisitionofthe autotrophicgrowth.Nearlyhalfthegenome(984genesoutof
electrondonor,incontrasttomostaerobicmicrobialfermen- 2293) responded with transcriptional changes of 2-fold or
tationswhereacquisitionofthefinalelectronacceptor,oxygen, greaterwhencomparingheterotrophytocarbondioxide-lim-
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ActivityofbothpurifiedMsed_0394andMsed_0406on4HB
waswellabovethereportedactivitymeasuredinautotrophic
cell extract (0.3 (cid:4)mol min(cid:5)1 mg(cid:5)1) (4). It appears that
Msed_0406 is primarily a promiscuous propionate-CoA syn-
thetase. Msed_0394, by contrast, has nearly equal levels of
activityonacetate,propionate,and4-HB.Althoughtheoverall
activity for Msed_0394 is lower by comparison, when taking
into account the different substrate specificities, this enzyme
shows a preference for C5-C6 linear unsubstituted organic
acids. By comparison, the homologous 4-HB-CoA synthetase
fromT.neutrophilus(Tneu_0420),ananaerobicarchaeonthat
containstheDC/4HBcarbonfixationcycle,wasrecombinantly
producedandshowntohavemaximalactivityon4HB,followed
bycrotonate,acetate,3HP,and3HB(9).ThereportedK value
m
for Tneu_0420 was about 3-fold lower than that found for
Msed_0406(700(cid:4)Mversus2000(cid:4)M),withcomparableactivity
(1.6versus1.8(cid:4)molmin(cid:5)1mg(cid:5)1),whichsuggeststhatthecat-
alyticactivitieson4HBarealsocomparable.
FIGURE 6. Reaction rate profile for acyl-CoA ligases. Michaelis-Menten
reactionratecurvesshownwithexperimentaldataforMsed_0394(squares), ItislikelythatMsed_0406ismoreeffectiveatcatalyzingthe
Msed_0406(circles),andMsed_1353-G424(triangles)overarangeofsub- ligationofCoAto4HBinvivothanMsed_0394.Perhapsthese
strateconcentrations.
enzymeshaveevolvedfromhighlyspecificacetate/propionate
synthetasestobesufficientforcatalyzingthenecessaryreaction
itedautotrophy.Notmuchisknownabouttheregulationstrat- on4HBforthe3HP/4HBfixationcycle.Itisnotclearwhytwo
egies employed by archaea to control gene transcription, but synthetases would be required, or whether both of them are
between different forms of chemolithoautotrophy (reduced necessaryforautotrophicgrowth.However,theyaresofarthe
metals,H ,etc.)andheterotrophy,M.sedulacanutilizeabroad onlyligasesinM.sedulathathavebeenshowntoactivate4HB
2
rangeofmetabolicsubstratesforgrowth. withCoA.
Themissingstepinthe3HP/4HBpathwayhasbeentheacyl- Genes with high homology to Msed_0394 and Msed_0406
CoAsynthetasethatutilizes4HB.Previousattemptstoidentify existinthegenomeofthecloselyrelatedM.cuprina(67and
thegenethatencodesthisenzymewereunsuccessful,andthe 73% amino acid identity, respectively), but it is less clear
candidateenzymeshadnoactivityon4HB(9).Inthiswork,two whetherhomologsexistinthegenomesofotherSulfolobales,
previouslyunexaminedsynthetasesfromM.sedula,consistent suchastheSulfolobusandAcidianusspp.Membersoftheacyl-
with the new transcriptomic evidence, were recombinantly adenylate-forming enzyme family may share little identity or
producedandcharacterized.BothMsed_0394andMsed_0406 similarityinaminoacidsequenceapartfromafewhighlycon-
showed activity on 4HB as well as other small organic acids. servedcoremotifs(27).TherearehomologsofMsed_0406in
Basedonthelackofothersynthetasecandidatessuggestedby otherspeciesofSulfolobalesthathave30–35%identityandone
thetranscriptomicanalysisandpreviousbiochemicalevidence homologinS.acidocaldariuswith61%identity.Buttheeffort
rulingoutMsed_1422andMsed_1291,weconcludethatoneor tofindtheM.sedula4HB-CoAsynthetasehasshownthatsub-
bothoftheseenzymesarenecessaryforautotrophicgrowthin stratespecificitycannotbeinferredfromaminoacidsequence
M.sedula. homologyalone.However,thelowhomologyoftheM.sedula
Acetyl-CoAsynthetasesbelongtotheclassIsuperfamilyof 4HB-CoAsynthetasegenedoesstandoutamongalltheother
adenylate-forming enzymes that includes acyl- and aryl-CoA genesinthe3HP/4HBcycle,whichhavedistincthomologsin
synthetases,theadenylationdomainsofnonribosomalpeptide Sulfolobusspp.thatrangefrom50to80%identity.
synthetases, and firefly luciferase (23). These enzymes use a Because4HBisametaboliteuniquetobutyratemetabolism
two-stepmechanisminwhichfirstanacyl-AMPintermediate (28),including(cid:3)-aminobutyratefermentation(29)andpolyhy-
isformed(withreleaseofpyrophosphate)followedbydisplace- droxyalkanoateproduction(30),itisunlikelytohaveanyother
mentofAMPbyCoA(24).Mostacetyl-CoAsynthetaseshavea role in crenarchaeal metabolism outside of carbon fixation.
limitedsubstraterange.Archaealacyl-CoAsynthetases,which Recent work with metabolic flux analysis has shown there is
formaphylogeneticclusterdistinctfromotherbacterialsub- anotherexitrouteforcarbonfluxfromthecyclethroughsuc-
groups (25), have been reported to exhibit broader substrate cinyl-CoAtosuccinate(10).Inthisstudytheauthorsestimate
preferences. The acetyl-CoA synthetase from Pyrobaculum thattwo-thirdsofthecyclecarbonfluxpassestosuccinatevia
aerophilum can work on acetate, propionate, butyrate, and succinyl-CoAorsuccinicsemialdehyde,althoughone-thirdof
isobutyrate(26);anotheracetyl-CoAsynthetasefromArchaeo- thecyclecarbonfluxpassesthroughthelatterpartofthecycle
globusfulgiduswasactiveonacetate,propionate,andbutyrate (via4HB)toregenerateacetyl-CoA.Ofcourse,thisfluxdistri-
(27). Both Msed_0394 and Msed_0406 were found to have bution may be highly dependent on growth conditions and
activityonabroadrangeofsmallorganicacidsubstratesofup could shift more to the 4HB branch depending on substrate
tofivecarbonsinlength. availability.
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FIGURE7.S.entericaACSandMsed_0394activesitecomparison.ACSisshowningoldandMsed_0394incyan.LigandfromACSstructure(adenosine-5(cid:4)-
propylphosphate)shownwithcoloredheteroatoms.A,sideviewofbindingpocketwithinter-atomicdistancesgivenfromphosphorusatomofpropyl-
phosphatemoietytoselectatomfromaminoacidresidues.B,axialviewfrombottomofsubstratebindingpocket.
ItisclearthatallmembersoftheSulfolobalesorderhavea
homologfor4hbdandthereforeshouldhaveacompletesetof
enzymes for carbon fixation. However, previous studies have
been mixed as to which Sulfolobus spp. are capable of
autotrophicgrowth.EarlyreportsonSulfolobusacidocaldarius
isolates claimed that they could grow chemolithoautotrophi-
callyonelementalsulfur(31,32).Subsequentreportsclaimthat
neither S. solfataricus nor S. acidocaldarius can grow
FIGURE 8. Sequence alignment of S. enterica acetyl-CoA synthetase
autotrophically on elemental sulfur alone (33), although it is (STM4275)andM.sedulaacyl-CoAligases.Aminoacidsequencealign-
unclear whether they simply lost the ability to grow chemo- mentofactivesiteresiduesinputativeacyl-CoAligasesrevealsaconserved
glycine(showninred)exceptforMsed_1353,whichhasatryptophanindic-
lithoautotrophicallyorwereselectedfromwhatwereoriginally ativeofacetate-propionateCoAligases.AlignmentwasgeneratedusingChi-
mixed cultures (34). Recent reports have shown autotrophic merabysuperpositionofI-TASSERthree-dimensionalstructuralmodels.
growthofSulfolobusmetallicusonsulfurandSulfolobustoko-
daiionbothsulfurandiron(35).Theonlyothermemberofthe thereforeonlyworkonacetateandpropionate,afactthathas
Sulfolobales that has been reported to grow on hydrogen is beenconfirmedbiochemically(9).Here,therewassomeactiv-
Acidianusambivalens,asulfur-reducingacidophile(36).Genes ity with Msed_1353 on 3HP and butyrate but no activity on
encoding for hydrogenase and maturation enzymes with 4HB. Msed_0394 and Msed_0406 both have a glycine in this
homologytoM.sedulahydrogenasegenesarepresentinone position,Gly333andGly346,respectively.However,therestof
strainofSulfolobusislandicus(HVE10/4),butthisispredicted thegenesannotatedasacyl-CoAsynthetasesinM.sedulaalso
to be involved in anaerobic fermentation (37). Clearly, some haveaglycineinthisposition,sothisglycineresiduealoneis
Sulfolobusspp.musthaveafunctionalcarbonfixationpathway, notsufficienttoindicateactivityonC3-C5unsubstitutedlinear
but others seem to possess an incomplete or nonfunctional organicacids.BothMsed_1422andMsed_1291wererecombi-
pathway.ItmaybethattheCoA-activatingligasethatcanoper- nantly expressed and shown to be inactive on C2-C4 linear
ateon4HBisessentialforcompletecyclefunction,andlossof organicacids(9).
4HB-CoAsynthetaseactivityrendersthecarbonfixationcycle AmutantofMsed_1353withaglycineinplaceofthecon-
inoperable. served tryptophan (W424G) was made by site-directed
Toinvestigatetheissueofsubstratespecificity,denovostruc- mutagenesisandexpressedinE.coli(Msed_1353-Gly424).The
turalpredictionsofM.sedulaacyl-CoAsynthetaseswerecom- nativeenzymewasactiveonlyonacetateandpropionate,but
pared with crystal structures of other known synthetases, themutantshowedactivityon3HP,4HB,valerate,hexanoate,
includingacetyl-CoAsynthetasefrombothS.enterica(17)and and even octanoate (Fig. 5). The activity was just as high on
Saccharomyces cerevisiae (38) and 4-chlorobenzonate-CoA C5-C8 substrates as on acetate and propionate but lower on
synthetase from Alcaligenes sp. (39). The structure for ACS 3HP and 4HB. This suggests that the polar hydroxyl group
fromS.entericarevealedthattherearefourresiduesthatform destabilizestheinteractionbetweenthesubstrateandtheresi-
theacetatebindingpocketasfollows:Val310,Thr311,Val386,and duesoftheenlargedbindingpocket.Asimilartrendisevident
Trp414(17).Theconservedtryptophanresiduecutsthebinding withMsed_0406(Fig.4).However,Msed_0394hasnearlyequal
pocketshortandprecludesactivityonlongersubstrates(Fig.7). levelsofactivityonpropionate,butyrate,and4HB,suggesting
ExtensivemutagenesisofbindingpocketresiduesinyeastACS thatitcanstabilizethehydroxylgroupon4HBbetterthanthat
showedthatmutationofW416Gwassufficienttolengthenthe of3HP.Similarly,Msed_1456,whichcatalyzestheligationof
binding pocket to accommodate C4-C8 organic acids (40). CoA to 3HP in the 3HP/4HB pathway, has equal activity on
AminoacidsequencealignmentsshowthatMsed_1353hasa propionateand3HP,andthereforeitmighthaveresiduesinthe
tryptophan in the same position (Trp424) (Fig. 8) and should activesitethathelpstabilizethehydroxylgroupof3HP.
4020 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER6•FEBRUARY8,2013
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InMsed_1456,Val386,whichmakescontactswiththe(cid:3)-car- 13. Han,Y.,Hawkins,A.S.,Adams,M.W.,andKelly,R.M.(2012)Epimerase
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replacedwithAsn390,whosepolaramidenitrogencouldhydro- malonyl-CoAtosuccinyl-CoAintheMetallosphaerasedula3-hydroxy-
propionate/4-hydroxybutyrate cycle. Appl. Environ. Microbiol. 78,
genbondwiththehydroxylgroupof3HPtostabilizesubstrate
6196–6202
binding.AsforMsed_0406,bothvalineresiduesintheacetate
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binding pocket are replaced with alanine (Ala249 and Ala321) man’sreagent.MethodsEnzymol.91,49–60
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threeoftheseresiduesarealanine(Ala240,Ala241,andAla309). electron transport chains in the extremely thermoacidophilic crenar-
chaeonMetallosphaerasedulathroughironandsulfurcompoundoxida-
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