Dodecin Is the Key Player in Flavin Homeostasis of Archaea

31 Pages·2009·1.98 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.284,NO.19,pp.13068–13076,May8,2009
©2009byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Dodecin Is the Key Player in Flavin Homeostasis of Archaea□S
Receivedforpublication,October21,2008,andinrevisedform,January9,2009 Published,JBCPapersinPress,February17,2009,DOI10.1074/jbc.M808063200
MartinGrininger‡1,HeikeStaudt§,PatrikJohansson‡,JosefWachtveitl§,andDieterOesterhelt‡2
Fromthe‡DepartmentofMembraneBiochemistry,MaxPlanckInstituteofBiochemistry,AmKlopferspitz18,82152Martinsried
andthe§InstituteofPhysicalandTheoreticalChemistry,GoetheUniversityFrankfurtamMain,Max-von-Laue-Strasse7,
60438FrankfurtamMain,Germany
Flavinsareemployedtotransformphysicalinputintobiolog- ically active compound has not been reported to date (1);
icaloutputsignals.Inthisfunction,flavinscatalyzeavarietyof instead, RF acts as the direct precursor for FMN and FAD in
light-inducedreactionsandredoxprocesses.However,nature flavinbiosynthesis.
also provides flavoproteins with the ability to uncouple the Inallflavins,thecatalyticallyactiveunitistheisoalloxazine
mediationofsignals.Suchproteinsaretheriboflavin-binding substructure(2–4).Tohandlethereactivityoftheisoalloxazine
proteins(RfBPs)withtheirfunctiontostoreriboflavinforfast ring,FMNandFADaretightlyboundtoflavoenzymes.Finely
deliveryofFMNandFAD.Herewepresentinvitroandinvivo tunedbindingrestrictsthereactionspectrumofFMNandFAD
datashowingthattherecentlydiscoveredarchaealdodecinisan to discrete, beneficial reaction pathways, preventing harmful
RfBP,andwerevealthatriboflavinstorageisnotrestrictedto side reactions. Reported functions of flavoenzymes include
eukaryotes. However, the function of the prokaryotic RfBP transferringelectronsfromandtoreactivecenters(e.g.respira-
dodecinseemstobeadaptedtotherequirementofamonocel- torychain)aswellasemployinglightfortheinductionofradi-
lularorganism.WhileineukaryotesRfBPsareinvolvedintraf- calreactions(e.g.DNA-photolyase)orconformationalchanges
ficking riboflavin, and dodecin is responsible for the flavin (e.g. phototropin) (5–8). In the biosynthesis, the full catalytic
homeostasisofthecell.Althoughonly68aminoacidsinlength, powerofflavinshasalreadyformedatthestageoftheFMNand
dodecinisofhighfunctionalversatilityinneutralizingribofla- FADprecursorRF(seeFig.1).AsRFisnotusedasanenzymat-
vintoprotectthecellularenvironmentfromuncontrolledflavin ically active compound, it is, different to FMN and FAD, not
reactivity.Besidesthepredominantultrafastquenchingofexcited integrated into flavoenzymes. To avoid a negative impact of
states,dodecinpreventslight-inducedriboflavinreactivitybythe uncontrolled flavin reactivity by free RF, nature established
selective degradation of riboflavin to lumichrome. Coordinated sequestering devices termed riboflavin-binding proteins
with the high affinity for lumichrome, the directed degradation (RfBPs).Theseproteinshavebeenshowntostoreanddistrib-
reactionisneutraltothecellularenvironmentandprovidesan uteRFineukaryotes,ensuringafastsupplyofFMNandFAD
alternative pathway for suppressing uncontrolled riboflavin (9,10).
reactivity.Intriguingly,thedifferentstructuralandfunctional ThesmallflavoproteindodecinfromthearchaeonHalobac-
propertiesofahomologousbacterialdodecinsuggestthatdode- terium salinarum was reported to bind RF with high affinity
cinhasdifferentrolesindifferentkingdomsoflife. andtoextensivelyquenchflavinfluorescence,thusexertingthe
keyfunctionsofRfBPs(11).Additionalaffinityforlumichrome
(LCR)couldmoreovercharacterizedodecinasaproteinwitha
Flavinsareamajorclassofcofactorsthatareabletoaccept dualbindingmode(12–14).Basedonsequenceconservation,
anddonateelectronsaswellastoabsorbvisiblelight.Flavins dodecinsclusterinanarchaealandabacterialsubgroup.The
consistofanonconservedaliphaticmoiety,covalentlylinkedto majorityofdodecinsoccurinbacteria,someamongpathogenic
aconservedaromaticisoalloxazinering(Fig.1andsupplemen- organisms such as Mycobacterium tuberculosis or Pseudomo-
talFig.1).Thedistributionofflavinsinnaturecorrelateswith nas aeruginosa. Here we present data that identify dodecin
the complexity of the aliphatic chain. Although FMN (phos- from H. salinarum as an RfBP. By storing RF in micromolar
phoribitylchain)andFAD(ADP-ribityl)arewidespreadamong amounts during growth-limiting conditions, and providing it
flavoenzymes,afunctionofriboflavin(RF,3ribityl)asacatalyt- forthebiosynthesisofFMNandFADathighmetabolicactivi-
ties, the archaeal dodecin acts as an RF buffer, balancing the
total flavin concentration during the H. salinarum life cycle.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains Analysisofthe40%sequence-identicalbacterialdodecinfrom
additionalExperimentalProcedures,Figs.1–8,Tables1–3,andadditional H.halophilarevealsdifferentstructuralandfunctionalproper-
references.
tiesandexcludesanRFbindingandstoringfunction.Although
Theatomiccoordinatesandstructurefactors(codes2vxaand2vx9)havebeen
depositedintheProteinDataBank,ResearchCollaboratoryforStructural thearchaealdodecinrepresentsthefirstRfBPdescribedinpro-
Bioinformatics,RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/). karyotes,theroleofthebacterialdodecinremainsspeculative.
1Towhomcorrespondencemaybeaddressed.Tel.:[phone];Fax:
49-89-8578-3557;E-mail:[email protected]. EXPERIMENTALPROCEDURES
2Towhomcorrespondencemaybeaddressed.Tel.:[phone];Fax:
49-89-8578-3557;E-mail:[email protected]. Preparation of Vectors and Strains and Purification of
3Theabbreviationsusedare:RF,riboflavin;RfBP,riboflavin-bindingprotein; Proteins—Cloning, purification, refolding, and reconstitution
HsDod,archaealH.salinarumdodecin(ligandcompositionnotspecified);
of proteins HhDodB, HsDodA, and HsDodA;E45A were per-
HhDod,bacterialH.halophiladodecin(ligandcompositionnotspecified);
LCR,lumichrome;RT,realtime;TdDod,bacterialT.thermophilusdodecin. formedasdescribedearlier(13).H.salinarumstrainsR1(cid:1)dod
This is an Open Access article under the CC BY license.
13068 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER19•MAY8,2009
FunctionoftheArchaealDodecin
treated with acetonitrile. Nickel-chelating chromatography
was used for determination of ligands bound to His-tagged
dodecin.
Crystallization of HhDodB and HsDodA;E45A—Diffracting
crystalsofHhDodBgrewin0.4MNH H PO and0.1Mectoinat
4 2 4
18°Cbythevapordiffusiontechnique.HsDodA;E45Awascrys-
tallizedunderidenticalconditionsasHsDodA(13).Thestruc-
tureofHhDodBwassolvedbymolecularreplacementmethods,
using the atomic coordinates of HsDodA (Protein Data Bank
code2ccb)asasearchmodel.
For detailed information see supplemental Experimental
Procedures.
RESULTS
Structural Comparison of Dodecin from H. salinarum
(HsDodA)andH.halophila(HhDodB)—Thecrystalstructureof
dodecin from H. salinarum (HsDodA; superscript A for
archaeal)revealedahighlysymmetric,hollow-sphericalshaped
protein with an outer diameter of about 65 Å. The 7.4-kDa
monomers of simple (cid:1)1(cid:2)(cid:1)2(cid:1)3 topology are assembled into
dodecamerscomposedofa(cid:1)-sheetcorpuswiththe(cid:2)-helices
FIGURE1.Flavinbiosynthesis.GTPandribulose5-phosphatearetheeducts exposed on the outside. Binding pockets for dimers of flavin
inflavinsynthesis.Thekeystepinflavinbiosynthesisisthedismutasereac- andflavin-likeligandsarelocatedbetweenthetrimersubstruc-
tioncatalyzedbyriboflavinsynthase(a),buildingthetricyclicheteronuclear
tures of the dodecameric assembly. Crystal structures of
organicring isoalloxazine.RF isfurtherconvertedintoFMNbyriboflavin
kinase(b)andsubsequentlytransformedintoFADbyFADsynthase(c).The HsDodAwiththeligandsLCR,lumiflavin,RF,FMN,andFAD
catalyticallyactiveisoalloxazineishighlightedbyagrayrectangleintheRF
allowed the analysis of the dodecin-binding site in detail.
chemicalstructure.Foramoredetaileddescriptionoftheflavinbiosynthesis
seesupplementalFig.1. HsDodAbindsRF(KDof36nM)andthesmallligandLCR(10
nM)withhighaffinitybyusingslightlydifferentbindingmodes
and R1dodHis(cid:2) are derivatives of the H. salinarum strain R1 (13).Whentheligandbindingpropertiesofahomologouspro-
(DSM671)andweregeneratedbyablue-redselectionstrategy, tein from Halorhodospira halophila (HhDodB, superscript B
based on the PMKK100 vector (15), and on the base of the forbacterial)wereanalyzed,theaffinitiesforRFandLCRwere
pBPH-Mvector,respectively(16). foundtobesignificantlylower(KDof2.5and20.4(cid:3)M,respec-
Light Stability of Riboflavin and Spectroscopic Characteriza- tively) as compared with HsDodA. To investigate the differ-
tion—ForthedeterminationofRFstability,freeanddodecin- encesbetweenthearchaealHsDodAanditsbacterialHhDodB
boundRFwereilluminatedwithahighpressuremercurylamp counterpart,thecrystalstructureofHhDodBwasdetermined
at800milliwatt/cm2radiationenergy.A3-mmKG1filterwas toaresolutionof2.6Å(seesupplementalTable1).
usedtoabsorblightofshort((cid:3)300nm)andlong((cid:4)700nm) Inlinewiththeoverall40%aminoacidsequenceidentity,a
wavelength from the light source. The decomposition was structural comparison between the archaeal HsDodA and the
monitored with absorption spectroscopy and fluorescence bacterialHhDodBrevealedastrongconservationofthedode-
detectionofchromatographicallyseparatedsamples.Oxygen- cinmonomerfold,withanoverallrootmeansquaredeviation
freeconditionswereachievedbyseveralcyclesofdegassingthe of 0.65 Å (59 C-(cid:2)atoms), as well as the conservation of the
reaction solution under argon atmosphere. Femtosecond dodecamerichollowsphericalassembly(Fig.2,AandB).How-
pump/probe experiments were performed as described else- ever,despitetheirhighsimilarity,thearchitectureoftheflavin-
where(17). bindingsiteandtheflavin-bindingmodeofHhDodBwasstrik-
Western Blotting and RT-PCR—For determination of the ingly different compared with HsDodA. Whereas HsDodA
dodecinexpressionprofile,samplesoflysates(volumesofsam- binds dimers of flavins with the re-sides of the isoalloxazine
plesrelatedtotheopticaldensityofthecellculture)weresep- rings associated, HhDodB incorporated RF via si-si stacking
arated using SDS-PAGE and immobilized on polyvinylidene (Fig.2C).
difluoridemembranes.Dodecinwasdetectedusingpolyclonal AsshowninFig.2D,thereducedsizeoftheflavin-binding
IgY anti-dodecin antibodies. RT-PCR was performed as pocket due to the altered main chain conformation of the
describedelsewhere(18). nearby(cid:1)2strandgivesanexplanationoftheinvertedbinding
Quantitative Analysis of Riboflavin and Lumichrome—For modeofHhDodB.ThebackboneconformationoftheThr45–
analysisoftheintracellularflavinconcentrations,acelllysateof Ile49peptidestretchisnotonlycausedbychangesintheimme-
H.salinarumwastreatedwithethanolat80°C.Aftercentrifu- diate HhDodB sequence. In the HhDodB structures, a pheny-
gation,thesupernatantwaschromatographicallyseparatedon lalaninesidechainofaneighboringdodecinmonomer(Phe40)
areversedphasecolumn,andflavinsweremonitoredbysimul- wedges between (cid:1)2 and the amphipathic helix, forming an
taneousdetectionatwavelengthcouples381/464and450/520 edge-face interaction to a highly conserved aromatic residue
nm.ForanalysisofLCR,asampleofthelysatewasadditionally (Trp57).Themainchainshiftnarrowsthebindingpocketsof
MAY8,2009•VOLUME284•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13069
FunctionoftheArchaealDodecin
FIGURE2.ComparisonofHsDodAandHhDodB.A,superpositionoftheC-(cid:2)tracesofHsDodA(red)andHhDodB(blue)asseparatemonomersandasmonomers
partofthedodecamericassembly(superpositionontheHsDodAC-(cid:2)traceingraywithboundriboflavinsincolorcodeofmonomers).View1showsdodecin
orientedalonga3-foldaxisandview2alonga2-foldaxis.HsDodAlabelsareinblackandHhDodBingray.B,structure-basedsequencealignmentandC-(cid:2)root
meansquaredeviationbetweenthearchaealHsDodAandthebacterialHhDodB.ThelargeststructuraldeviationsinC-(cid:2)atomsbetweenHsDodAandHhDodB
involveresidues47–52(50–55ofHhDodB).Thispeakcorrespondstodifferencesintheloopbetweenthe(cid:1)2and(cid:1)3strands.C,invertedbindingofRFinHsDodA
(red)andinHhDodB(blue).Trp36(W36)andGln55(Q55),clampingandaligningtheisoalloxazineringsintetradearrangements,areshown(HsDodAnumbering).
IncontrasttoHsDodA,theglutamineinHhDodBbindstheflippedflavinofthedimer-relatedmonomer.Inbothstructures,thedistancebetweentheindoleand
theisoalloxazinearomaticringsisabout3.3Å.D,deviationinthemainchainarchitecturetomeetthedifferentrequirementsofligandincorporation.The
widenedpocketofHsDodAallowsaccommodationofthedimethylatedbenzeneringinthere-rearrangedaromatictetrade(seeminorpeakinFig.2Bbetween
residues42and46).Arg46(R46)ofHhDodB,highlyconservedinbacterialdodecins,accountsforhydrogenbondingtotheisoalloxazinemoiety.Thisisoallox-
azine-proteininteractionismissinginHsDodA.Forclarity,sidechainsofHsDodAareomitted,andthebindingpocketisreducedtothe2-foldrelatedpart.
HhDodB structure, promoting the tilted conformation of the acteristicsduringillumination,supportingthatLCRisarather
si-si-stackedisoalloxazinemoietiesoverthere-re-stackedcon- stableendproductoflight-inducedRFdegradation(20).
formationseenintheHsDodA. Spectroscopic Analysis of Free and Dodecin-bound RF—To
Recently, FMN incorporation into the dodecin homolog analyze the quenching competence of dodecin, time-resolved
from the bacterial thermophile Thermus thermophilus UV-visible spectroscopy was carried out using RF-reconsti-
(TtDodB) was shown to be similar to RF binding to HhDodB tuted dodecins (RF/HsDodA and RF/HhDodB). The excited
(19).Mutationofarginine,correspondingtoArg46inHhDodB, statedynamicsofRFwasrecordedbyexcitingatthreedifferent
formingH-bondstotheC-4(cid:5)OandN-5oftheisoalloxazine, spectralpositions,inducingdifferentelectronictransitions,and
doesnotinfluencethemodeofFMNbinding.Supportingthe
providingdifferentexcessvibrationalenergies.Pulsescentered
impact of the main chain conformation of the (cid:1)2-strand,
atwavelengthsof476nm(S /S transition),388nm(S /S tran-
TtDodBnarrowsthebindingpocketandformsedge-faceinter- 0 1 0 2
sition, low energy wing), and 345 nm (S /S transition, high
actions(Phe39/Tyr56),similarlyasfoundinHhDodB. 0 2
energy wing) were used to excite RF. Typical pulse energies
Light-inducedDegradationofRF—Thelightstabilityoffree
wereabout100nJ.Probepulsesinthespectralrangeof450–
and dodecin-bound RF was determined by illuminating RF-
750nmweregeneratedinasapphirecrystalassinglefilament
dodecin complexes with light from a high pressure mercury
white light. Transient absorbance changes for the different
lamp.Absorptionspectrawererecordedatvarioustimepoints,
pump wavelengths did not show any significant differences,
and concentrations of RF and the degradation product LCR
whichsuggeststhattheS /S transitionisfasterthanthetime
weremonitoredbyfluorescencedetectionofchromatographi- 2 1
resolutionoftheexperiment.Discussionofthetimeresolved
callyseparatedsamples(Fig.3AandsupplementalFig.2,Aand
data is thus restricted to the 388 nm excitation, yielding data
B).Similartothefreecompound,HhDodB-boundRFdecom-
withthebestsignaltonoiseratio.
posedintovariousfluorescentdegradationproducts(seetraces
HhDodB_iiand_iiiinFig.3A),butthehalf-life(t )of32sfor As illustrated by the transient absorbance changes seen in
1⁄2 Fig.4A,freeanddodecin-boundRFshowedentirelydifferent
theunboundRFwas45-foldincreasedto24.7mininthebound
spectral and dynamic characteristics. (a) While the excited
state(Fig.3B).
RFincorporatedintoHsDodAshowedslightlylowerstabili- statelifetimeoffreeRFwaslongerthanthemaximumdelayof
zationasRFboundtoHhDodB(RF/HhDodB)(t of19.7min) theexperiment(1.5ns),boundRFdisplayedexcitedstatelife-
1⁄2
butincontrastwasefficientlytransformedintoLCR.Evenafter timesdramaticallyshortenedtoafewpicoseconds.(b)Thepat-
4hofillumination,noothercompoundwasdetectedatboth ternofstimulatedemission(SE,blue),groundstatebleach(GB,
wavelengthcouples(seetracesHsDodA_iiand_iiiinFig.3A). blue) and excited state absorption (ESA, red) was strongly
AsdepictedinFig.3C,74%oftheinitialRFcouldbedetectedas changed,ase.g.theextensiveSEat520–600nmobservedfor
eithertheLCRproduct((cid:6)67%)orintactRF((cid:6)7%).Freeand free RF in aqueous solution was missing in both RF-dodecin
dodecin-boundLCRdidnotshowachangeinabsorptionchar- complexes(21).
13070 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER19•MAY8,2009
FunctionoftheArchaealDodecin
FIGURE3.Light-induceddegradationoffreeanddodecin-boundRF.A,illuminationoffreeanddodecin-boundRF.ChromatographicprofilesofRF/HsDodA
(HsDodA_i-HsDodA_iii)andRF/HhDodB(HhDodB_i-HhDodB_iii)at0minandafter4hofilluminationwithlight.Fluorescencedetectionisperformedat520(i,ii)
and464nm(iii).ChemicalstructuresforRF(E)andLCR((cid:2))areshownasinsetsinfluorescencelanes.NotethattheEUscaleisdifferentinlanesi–iii.B,molar
concentrationsofRFfittedtoafirstorderdecay;HsDodA(blackcircle,solidline)andHhDodB(graycircle,graysolidline)andfreeRFshownininset.Half-liveswere
determinedtobe32(cid:7)1.4s(freeRF),1180(cid:7)180s(19.7min;HsDodA)and1480(cid:7)230s(24.7min,HhDodB),respectively.C,lightstabilityofdodecin-bound
RF(inair).ConcentrationsofRFandLCRincorporatedintoHsDodA(F/f)andHhDodB(E/(cid:2))andtherespectiveoverallconcentrations(Œ/(cid:1))areshown.
However,differencesinspectrawerealsoobservedbetween LCRConcentrationsduringH.salinarumGrowth—Inparal-
RFboundtoHsDodAandHhDodB.(a)Thebroadexcitedstate leltothequantitativeanalysisofflavinsalsotheLCRcellular
absorptionsignalofRF/HsDodAdidnotextendintothelong concentrationsweredeterminedduringH.salinarumgrowth.
wavelengthregion,asseenforthebacterialRF/HhDodBaswell AsshowninFig.5C,concentrationsatday4werelowincellsof
asforfreeRF.(b)ExcitedstatelifetimesofRFboundtoHhDodB thewildtypeandthedodecindeletionstrain.However,starting
wereslightlylongerthanofRFboundtoHsDodA.Respective atday6,LCRconcentrationsinthewildtypecellsculturedin
individualtransientsareshowninFig.4B. light(R1_l)wereincreasedtoabout20(cid:3)M,whereascellscul-
FlavinConcentrationsduringH.salinarumGrowth—Flavins turedinthedark(R1_dandR1(cid:1)dod_d)keptLCRmolaritiesat
were extracted from H. salinarum R1 (wild type) strain cells, lowlevelsof5.4(cid:3)Mathighest(R1_d,day35,seealsosupple-
chromatographically separated, and quantified by fluores- mentalTable3).
cence.Recordedpeakareaswerecorrelatedtotheinternalcell Ligand Analysis of Homologously Expressed HsDodA—The
volume, calculated from the optical density of the cultures strain R1dodHis(cid:2) homologously expresses His-tagged dode-
(OD ) at the respective day (Fig. 5A). The following strains cin.AfterextractionfromtheH.salinarumcytosolbyanickel-
578
andcultureconditionswereexamined:H.salinarumwildtype chelating resin, ligands bound to His-tagged dodecin in vivo
straininthelight(R1_l)andinthedark(R1_d),andadodecin- werequantitativelydeterminedbychromatographicseparation
deficientstraininthedark(R1(cid:1)dod_d).Cellsofthewildtype and fluorescence detection. No FMN and FAD could be
strain,cultivatedinthedark(R1_d)aswellasinthelight(R1_l), detectedasnaturalligands.AspresentedinTable1,valuesfor
showed much higher FMN and FAD concentrations in the theRFcellularconcentrationsmeasuredattheendoftheexpo-
exponentialgrowthphase(day4and6)thaninthestationary nentialgrowthphase(day7),inthemiddle(day13),andthelate
phase(Fig.5B).Incontrast,RFconcentrationswerelowinthe stationary phase (day 21) correspond well to values of the H.
exponentialandearlystationaryphaseandhighinthelatesta- salinarumwildtypestrainR1(supplementalTable3).Concen-
tionary phase. RF regulation was most pronounced in R1_d trationsofdodecin-boundRFareintherangeofthetotalcel-
cells,wherevaluesreached18.2(cid:3)Minthelatestationaryphase lularRFandsuggestthatinH.salinarumallRFissequestered
(day23). bydodecin.
In the deletion strain R1(cid:1)dod, FMN and FAD concentra- ExpressionofDodecinduringH.salinarumGrowth—West-
tions resembled levels of the wild type strain R1. However, a ernblotanalysisofH.salinarumculturesrevealedthatdodecin
dramaticinfluenceofthedeleteddodecincouldbeseeninthe isregulatedduringgrowth.AsseeninFig.6A,dodecinexpres-
concentrations of RF; 18.6 (cid:3)M in the wild type inoculation sionwasgenerallyinducedupontransitionintotheexponential
culture grown in the dark (R1_d) is opposed by 1.2 (cid:3)M in growthphase,irrespectiveofthepresenceoflight.Afterinduc-
the corresponding dodecin-deficient R1(cid:1)dod pre-culture tion of dodecin expression, the protein level varied slightly
(R1(cid:1)dod_d).Thisdifferencewassignificantlydecreasedatday underbothconditions.ThecopynumberofdodecininanR1_d
4butappearedagainwhentheculturesranintothelatestation- cultureatthestationaryphasewasdeterminedto8000,corre-
aryphase(seealsosupplementalTable2). spondingtoaconcentrationof9.2(cid:3)M(seesupplementalFig.
MAY8,2009•VOLUME284•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13071
FunctionoftheArchaealDodecin
LCRConcentrationsunderLaboratoryConditionsandinthe
Native Environment—Table 2 lists the LCR concentration in
non-inoculatedfreshmedium,eitherstoredinlightorindark
forseveralweeks,andinwaterstakenfromhypersalineponds,
an environment where Haloarchaea occur in high numbers.
WhereasintheH.salinarumgrowthmediumLCRwasfound
to be present in high nano- to low micromolar amounts, the
concentrationofLCRinthenaturalenvironmentwas102–103-
foldlower.
DISCUSSION
Dodecin Buffers the Flavin Concentration during H. salina-
rum Growth—The analysis of the flavin metabolism revealed
thatcellularRFconcentrationsweresignificantlydependenton
dodecin. As illustrated in Fig. 5B, the RF level was 4–16-fold
higherinH.salinarumwildtypecells(R1_d)ascomparedwith
thedodecin-deficientstrain(R1(cid:1)dod_d),indicatingtheinflu-
enceofdodecin.Thenatureofdodecinasarigid,dodecameric
RF-bindingparticledoesnotsuggestadirectregulativerolein
flavin biosynthesis. The increased RF concentrations in wild
typecellsmostlikelyoccurfromindirectlyinfluencingthefla-
vinmetabolismbydepletingthecytosolofRFandsuppressing
a down-regulation of flavin synthesis (22–24). Data from RF
extractions of the dodecin overexpression strain R1dodHis(cid:2),
whichdemonstratedthattheallcellularRFcanbesequestered
bydodecin,areinlinewiththeassumptionthattheimpactof
dodecinontheflavinbiosynthesisresultsfromitscapacityto
storeRF(seeTable1).
ThebenefitofcreatingareservoirofRFisconnectedtothe
expression profile of dodecin and is illustrated in Fig. 7A.
Inducedduringexponentialgrowth(stage1),dodecinisconsti-
tutively expressed and stores RF, increasing in concentration
during the stationary phase (stage 2). If the environment
changestowardfavorablegrowthconditions,simulatedbyre-
inoculationintorichculturemedia,dodecinisdown-regulated
(seeFig.6A),andthecollectedRFsubsequentlybecomesavail-
ableforthesynthesisofFMNandFAD(stage3).Thisregula-
tion of dodecin enables a fast response of H. salinarum to
changingenvironmentalconditions.
FIGURE 4. Time-resolved spectroscopic characterization of free and WorkingMode1,UltrafastQuenchingofExcitedRF—X-ray
dodecin-boundRF.A,excitationofRF(paneli),RFincorporatedinHsDodA
structural investigations revealed that the isoalloxazine of
(RF/HsDodA)(panelii),andinHhDodB(RF/HhDodB)(paneliii)performedat
388nmwithsapphirewhitelightastheprobingpulse.Positiveabsorbance boundRFisdeeplyburiedintheproteininterior(seeFig.2D).
changes are shown in red and negative in blue. B, transient absorbance TheloopL3,connectingHsDodAstrands(cid:1)2and(cid:1)3,represents
changesat451nm.FreeRFisshownasadashedlineinblack,thecorrespond-
ing RF/HsDodA and RF/HhDodB complexes in black and gray solid lines, themostflexiblepartofthestructureandhasbeenproposedto
respectively. sealthebindingpocketandshieldtheisoalloxazineringfrom
theenvironment(13).Althoughprotectionoftheisoalloxazine
substructurefromchemicalactivationcaneasilybeachievedby
4A).RT-PCRwasemployedtoquantitativelymonitorthereg-
stericrestrictions,theprotectionfromlight-inducedreactions
ulationofdodecinatthemRNAlevelduringthetimeperiodof
ismoredemanding.
therelativelyconstantdodecinexpression.ThemRNAlevelsat
Thekeystepinflavinphotochemistryisthephotoexcitation
dayi((cid:1)Ct,xi)wererelatedtothestartinglevelatday4((cid:1)Ct,x1).
of the isoalloxazine electronic system into an excited singlet
Increasing values indicate that dodecin was slightly up-regu-
state,whichisgenerallyaccompaniedbyasubsequentintersys-
latedatthetranscriptionallevelduringgrowth(Fig.6B).Com- tem crossing into the triplet state. Excited singlet and triplet
parisonofdodecinmRNAlevelsincellsgrowninlight((cid:1)Ct,l) stateenergiescanbereleasedinintra-andintermolecularreac-
andinthedark((cid:1)Ct,d)revealedthatdodecinwasnotregulated tions,constitutingtheflavinphotochemistry.Althoughunable
bylightatthetranscriptionallevel.Foradiscussionoftheout- toinhibittheinitialstepofthephotoinducedformationofthe
lyingdodecinmRNAlevelsatday9,seesupplementalExperi- excited state, proteins can efficiently suppress light-induced
mentalProceduresandsupplementalFig.4B. reactivity. As has been shown for the chicken RfBP, binding
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FunctionoftheArchaealDodecin
FIGURE5.FlavinandLCRconcentrationsduringH.salinarumgrowth.A,growthcurvesofH.salinarumstrainsR1(wildtype)andR1(cid:1)dod(dodecindeleted).
Thecolorcodeofgrowthphases(exponential,yellow;earlystationary,orange;latestationary,red)isusedinBandC,andFig.7A.B,flavinconcentrationsduring
H.salinarumgrowth.QuantitativeanalysisofintracellularflavinrevealssimilarFMN(star)andFAD((cid:1))concentrationsinwildtypecellsculturedinthedark
(R1_d,black)andinthelight(R1_l,red),aswellasinthedeletionstrainR1(cid:1)dodinthedark(R1(cid:1)dod_d,gray).ConcentrationsofRF(E)showmajordifferences
withrespecttolightanddodecin.C,LCRconcentrationsduringH.salinarumgrowth.LCRmolarconcentrationsareabout10-foldincreasedintheH.salinarum
wildtypestrainR1growninlight(R1_l,red)ascomparedwithcellsgrowninthedark(R1_d,black)ordepletedindodecin(R1(cid:1)dod_d,gray).LCRconcentrations
werebelowthedetectionlimit((cid:3)0.5(cid:3)M)intheR1(cid:1)dod_dinoculationcultureandatday4inculturesR1_dandR1(cid:1)dod_d.
TABLE1 TABLE2
RFconcentrationsinculturesofR1dodHis(cid:1) LCRconcentrationsinH.salinarummediaandinthenaturalhabitat
Dark Light Natural
Day Freshmediuma Oldmediumb habitatc,d
BoundRF TotalRF BoundRF TotalRF
Dark Light Dark Light Sample1 Sample2
(cid:3) (cid:3)
M M
7 0.88(cid:7)0.12 1.50(cid:7)0.03 1.16(cid:7)0.05 1.11(cid:7)0.11 (cid:3)M (cid:3)M (cid:3)M
13 1.89(cid:7)0.05 1.57(cid:7)0.15 1.34(cid:7)0.27 1.16(cid:7)0.12 LCR 0.3(cid:7)0.02 2.1(cid:7)0.01 1.58(cid:7)0.1 0.17(cid:7)0.01 0.011 0.0034
21a 17.98(cid:7)1.25 19.38(cid:7)1.11 8.06(cid:7)0.91 11.57(cid:7)0.72
aFreshlypreparedmediawerekeptfor2daysinthedarkorinthelight.
aValuesweregeneratedinanindividualexperiment. bMediaofH.salinarumculturesweregrowntothelatestationaryphase.
cExtractionoftwoindependentwatersampleswerefromthesolarsalternsinEilat,
Israel.
dValuesweredeterminedbystandardadditionmethod.
Time-resolved spectroscopy performed on the dodecins
RF/HsDodA and RF/HhDodB recorded ground state recovery
within a few picoseconds, and thus demonstrates a similarly
efficient depopulation of RF excited states as reported for
chickenRfBP.Basedonthesimilararchitectureofthechicken
RfBPandthedodecinbindingpockets,wesuggestalsothatin
thedodecinsHsDodAandHhDodBtheindolemoietiesofthe
adjacent tryptophans transfer the electron for RF quenching.
Consideringthetryptophan-isoalloxazinedistancesof3.3Åin
the HsDodA and the HhDodB binding pockets, which are
shorter than the respective distances of tryptophan (Trp156)-
isoalloxazine(3.7Å)andtyrosine(Tyr75)-isoalloxazine(3.7Å)
inthechickenRfBP,electrontransfercanbeestimatedtotake
placewithinthesub-picosecondtimescale(26,27).
FIGURE6.DodecinproteinandmRNAlevelsduringgrowthofH.salina- Ithastobementionedthatthecurrentviewonthedodecin
rum.A,WesternblotanalysisofdodecininR1wildtypestraingrowninthe quenching characteristic is based on the extrapolation from
dark(R1_d)andinlight(R1_l).B,analysisofthedodecinmRNAlevelsincellsof
R1_d(f),R1_l(F,dashedline),andR1(cid:1)dod_d(graytriangle)inthetime similarproteins.Atpresent,itcanjustbespeculatedtowhich
period of the rather constant dodecin expression observed in A. A extent electronic interactions between the stacked RF dimers
2(cid:8)(cid:1)(cid:1)Ct(xi(cid:8)x4)valueof1meansnodifferenceinthedodecinmRNAlevelatthe
increasethecomplexityofthephotochemicalbehavior.
respectivedaycomparedwithreferenceday4;1⁄2and2,a2-folddown-
regulationandup-regulation,respectively.2(cid:8)(cid:1)(cid:1)Ct(l(cid:8)d)valuesillustratethe WorkingMode2,DirectedDegradationofExcitedRF—When
differencesindodecinmRNAlevelswhencellsgrowninlight(R1_l)andin incorporated into HsDodA, we found that RF was protected
dark(R1_d).
fromfastdegradationbutalsofromunspecificdecomposition.
WhereastheenhancedstabilityofRFistheresultoftheultra-
pockets, clamping the isoalloxazine ring between aromatic fast recovery of the RF ground state, the almost quantitative
moieties,canstronglymodifyRFphotochemistrybyefficiently transitionofRFtoLCRcouldberecognizedasthekeycharac-
shorteningthelifetimeoftheRFexcitedstate(11,25).Herein, teristicofanalternativeworkingmodecoupledtoRFstorage.
theindolemoietyofthetryptophan,holdingtheisoalloxazine Asthisdirecteddegradationiscoordinatedwiththehighaffin-
ringofRF,quenchestheexcitedstatelifetimebytheultrafast ityofdodecinforLCR,HsDodAallowsanoverallcompartmen-
transfer of electrons onto the excited flavin, creating a short- talizedreaction,whichisneutraltothecellularenvironment,
livedRF/tryptophanradicalpair. andestablishesanalternativepipelineforthedepopulationof
MAY8,2009•VOLUME284•NUMBER19 JOURNALOFBIOLOGICALCHEMISTRY 13073
FunctionoftheArchaealDodecin
FIGURE7.Workingschemeofthearchaealdodecinonthecellularandontheproteinlevel.A,dodecincycle.Dodecinexpressionisinducedinthe
exponentialphase.Althoughpresent,theuptakeofFMNandFADispreventedbytheirlowaffinity(stage1).Inthestationaryphase,RF(orange)from
degradationofflavinsand/orbiosynthesisissequesteredbydodecin,establishingaflavinbuffer(stage2).StoredRFisavailableforbiosynthesisofFMNand
FADwhenfavorableconditioninducedmetabolicactivity(earlyexponentialgrowthphase,stage3).Intracellularflavinconcentrationsaregivenininsets.
AttachedschemesabstracttheinterplayoffreeRF,boundRF(RF/HsDodA),andconvertedRF(FMN/FAD).B,workingmodesofHsDodA.Shownisamodelfor
thenondestructivequenching(workingmode1,green)andthedestructivedealkylationreaction(workingmode2,red)asparallelreactions.Photoreduction
andphotodealkylationarereportedtoleadtoLCRasadegradationproduct.BecauseofthehighaffinityofHsDodAforLCR,LCRiskeptcapturedinthebinding
pocket.Theintermediatestateinworkingmode1isshownasachargedradicalpairpriortoaputativeprotonation/deprotonationstep.
RFexcitedstates.Arationaleforinstallingadestructivedeal- the transition state. For a short description of RF binding in
kylationofRFasasecondreactionmightbethepossibilityto HsDodA;E45AandthedegradationofRFboundtoHsDodA;E45A
preventunspecificreactivityofRF,leadingtoreactive(degra- seethesupplementalmaterial.Thecoordinationoftheribityl
dation)speciesthatcouldnotbecontrolledbyproteinligand chaininfluencesthedegradationspectrum(seesupplemental
binding. The alternative working mode constitutes an emer- textandsupplementalFig.5,AandB).
gencypathwayforrelaxationoflight-activatedRF,whichmight As illustrated in Fig. 4A, transient absorption changes
beofparticularrelevanceforHaloarchaeathatgenerallyoccur recordedforRF/HsDodAandRF/HhDodBrevealedsignificant
inbrightlightenvironments.Recently,directedflavindegrada- differencesintheelectroniclandscapesoftherespectiveRFs.In
tionwasalsoreportedfortheFMN-bindingproteinBluBfrom particular,therecoveryoftheRFgroundstate,criticalforsup-
Sinorhizobiummeliloti.Incontrasttododecin,whichsacrifices pressing the RF light-induced reactivity, was faster for the
RF for the cellular integrity, BluB degrades FMN to deliver a archaeal dodecin. A satisfactory global fit analysis of the two
cobalaminbuildingblock(28,29). datasetsobtainstwotimeconstantssimilarforbothproteins
ReactionControlinDodecins—AsillustratedinFig.7B,non- (HsDodA,0.9and6.6ps;HhDodB,2and9.8ps).Theindividual
invasiveultrafastquenchingofRFexcitedstatesanddestruc- weights and the spectral characteristics in the corresponding
tive dealkylation are considered as parallel reactions down- amplitudespectraare,however,clearlydifferent,indicatingdif-
stream of the excited RF. Ultrafast quenching is the primary ferent reaction channels for excited state quenching in the
processandisresponsiblefortheincreasedlifetimeofdodecin- archaealandthebacterialdodecin.
boundRF. It cannot be stated that the spectral behavior is the only
Thehighselectivityofthedegradationreactionislikelysup- determinantoftheunspecificphotodegradationofRFboundto
portedbytheextensivedecreaseinRFexcitedstatelifetime,as HhDodB.ConsideringthelowaffinityofHhDodBforRF(K 2.5
D
onlyreactionsthatproceedwithinthenarrowtimewindowof (cid:3)Mversus36nMforRF/HsDodA),adecompositionofRFinthe
picosecondscancompetewithultrafastquenching.Suchcom- unboundstateofadynamicbindingrebindingprocesscannot
peting ultrafast processes require a reaction partner in close beruledout.
proximitythatisfulfilledfortheintramolecularreactionmode. DodecinWorkingMode2LeadstotheAccumulationofLCR—
Mechanistically,thestrictlyintramolecularphotodealkylation Although the catalytic properties of flavins are widely used,
and the intramolecular mode of photoreduction can be envi- only a few examples of the physiological importance of LCR
sionedasroutestolumichrome.Bothreactionswerereported havebeenreported(33,34).Instead,LCRisknownasatoxic
toproceedfromtheexcitedsingletstate,andthusinthetime substance,transferringlightenergytosubstrates(photosen-
range of the excited state lifetime of dodecin-bound RF (4). sitization type 1) and oxygen (photosensitization type 2),
Moreover, both pathways pass a six-membered cyclic transi- creating reactive compounds like singlet state oxygen (35).
tionstatethatispre-organizedinRFboundtoHsDodA(N1- Accordingly,wefoundasharpdecreaseofcelldensitywhen
C10a-N10-C1(cid:9)-C2(cid:9)-H2(cid:9)) and could well represent the struc- H.salinarumwasgrowninthelightandinthepresenceof40
turalbasefortheselectionofRFdealkylation(4,30–32).This (cid:3)M LCR. In the dark, LCR did not effect cell growth (see
viewissupportedbytheevenhigherselectivityofRFdealkyla- supplementalFig.3B).
tionintheE45Amutateddodecin(HsDodA;E45A).Thelackof InourexperimentsonH.salinarumcellsgrownunderlabo-
Glu45mediatedribitylchainbonding,andtheenhancedcon- ratory-rich conditions, we found intracellular concentrations
formationalflexibilitymightallowfine-tuningthegeometryof of LCR in the low micromolar range, even when cells were
13074 JOURNALOFBIOLOGICALCHEMISTRY VOLUME284•NUMBER19•MAY8,2009
FunctionoftheArchaealDodecin
haveadifferentarchitectureoftheirRF-bindingsites.Biophys-
ical analyses also indicated fundamentally different spectro-
scopic properties, and light degradation experiments showed
entirely different degradation spectra (see Figs. 3 and 4).
Although our data clearly suggest that the function of the
archaealdodecinistostoreRF,theroleofthebacterialdodecin
isnotclearatpresent.Thestructurallyandfunctionallychar-
acterizedbacterialdodecinsfromH.halophila(HhDodB)and
T.thermophilus(TtDodB)showbindingspectradifferentfrom
that of H. salinarum (HsDodA), supporting the flavins FMN
andFADinafunctionalrole(13,19).Recently,thecrystalstruc-
tureoftheNADH:FMNoxidoreductase(EmoB)hasbeenpub-
lished, revealing FMN dimers bound as si-si-stacked dimers,
similar as found in the bacterial dodecins (38). However,
FIGURE8.Ramppplotofdodecinhomologoussequences.107sequences whereas dodecin provides two identical binding positions
identifiedashomologsofHsDodAarelistedaccordingtostandardtaxonomy
deeply buried in the protein interior, EmoB binds the FMN
of the respective species and blasted against each other. E-values are
arrangedinatwo-dimensionalmatrixwithcolorsrepresentingtheconserva- molecules of a stacked dimer differently and exposed at the
tionaccordingtothelegendatleft.EachpixelrepresentstheE-valueofthe proteinsurface,promotingaping-pongmechanismincatalysis.
sequencepairattherespectivehorizontalandverticalaxis.Thearchaealsub-
familyclustersasahighlyhomologousgroupofsequences,withsignificantly Foramoredetailedviewonaputativefunctionofthebacterial
lowersimilaritytobacterialdodecins.Thisisindicatedbyadarkframewitha dodecinseesupplementaltextandsupplementalFig.8.
brightspotalongthediagonal(foralistoforganismseesupplementalFig.6
Concluding Remarks—We have shown that RF storage for
andFig.7).
fastdeliveryofFMNandFADisnotrestrictedtohigherorga-
growninthedarkwherelight-induceddegradationofRFwas nisms but also operates in prokaryotes. The function of
prevented. Analysis of non-inoculated H. salinarum growth HsDodA is to sequester RF under growth limitation and to
mediumrevealedLCRconcentrationsof0.3(cid:3)Minfreshand2.1 releasethiscompoundintothebiosynthesisofFMNandFAD,
(cid:3)Minlight-incubatedmedium(seeTable2),suggestingthatthe whenthemetabolicactivityisinducedbyfavorableconditions,
high levels of internal LCR are the result of the amphiphilic thusmaintaininganelevatedlevelofflavinthroughouttheH.
nature of LCR, which allows this compound to enter the cell salinarum life cycle. Because of the high reactivity of flavins,
interiorviapassivediffusionacrossthecellmembrane.Acon- storageofRFbearsariskforthecellularintegrity.Toprevent
centrationof200pM,asreportedforLCRinseawater(36),and uncontrolled reactions with potent molecules, HsDodA binds
concentrationsof2and10nM,asrecordedinsamplesfromthe RFwithhighaffinityandencapsulatestheisoalloxazineringsat
natural environment (see Table 2), imply that different from the bottom of binding pockets that are sealed with flexible
laboratory conditions H. salinarum is not exposed to high loops. Protection from light-induced reactivity is achieved by
exogenous levels of LCR under native conditions. The LCR ultrafastquenchingofRFexcitedstatelifetimes,putativelybya
bindingcapacityofdodecincanthereforeratherberegardedas similar pathway as has been described for eukaryotic RfBPs.
afunctionalelementthatactstokeepLCRsequestered,gener- Quenchingisassistedbyanemergencyrelaxationofdealkylat-
atedbythedodecinworkingmode. ingtheexcitedRFtoLCR.Bycoordinatingthisdirecteddegra-
While reflecting non-native conditions, tracing the LCR dationwiththeaffinityforLCR,thereactionproceedsinthe
concentrations under laboratory conditions illustrates how nanospaceoftheHsDodAbindingpocketandisneutraltothe
H.salinarumdealswiththeaccumulationofLCR.Evennon- cellularenvironment.
physiologicallyhighinternallevelsof12.8(cid:3)M,asdetectedin
theR1inoculationculture,weredecreasedwithinafewdays Acknowledgments—WeareverygratefultoUrselGrampp-Heider,
toconcentrationslowerthan0.5(cid:3)M(day4indark;1.8(cid:3)Min EvySauer,GaborMeser,andOliverSchmidtforassistanceinthe
light). This seems to be achieved by releasing LCR during laboratory;MarkusRamppforsupportinbioinformaticsondode-
down-regulationofdodecininearlyexponentialgrowthand cin; Georg Lentzen (Bitop, Germany) for providing ectoin; Frank
thedilutionofthediffusibleLCRtotheconcentrationlevel Siedlerfortheset-upofhighpressureliquidchromatographywith
of the habitat. Temporarily enhanced LCR concentrations fluorescencedetection;JerryEichlerandAharonOrenforsamples
from the solar salterns of the Israel Salt Co. (Eilat, Israel); Lissy
might thereby be tolerated in cells undergoing high meta-
Weyher-Stingl for analysis of dodecins by mass spectrometry;
bolicactivity.
Ehmke Pohl and Clemens Schulze-Briese (SLS, Zurich, Switzer-
DodecinsinArchaeaVersusDodecinsinBacteria—Asshown
land);andJoanneMcCarthyandDidierNurizzo(ESRF,Grenoble,
inFig.8,proteinalignmentsrevealthatdodecinsclusterinan
France) for beamline support. We also thank Mike Dyall-Smith,
archaeal and bacterial subgroup. Whereas widely distributed
LuisMoroder,AlfonsPenzkoferandPetraWollmannforreading
withinbacteria,archaealdodecinsareonlyfoundinthephylum
anddiscussingthemanuscript.
Haloarchaea(seealsosupplementalFigs.6and7).Weinitially
assumedthatthehaloarchaealdodecincladereflectstheadap-
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