Association of Novel Domain in Active Site of Archaic Hyperthermophilic Maltogenic Amylase from ...

12 Pages·2012·4.09 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.287,NO.11,pp.7979–7989,March9,2012
©2012byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Association of Novel Domain in Active Site of Archaic
Hyperthermophilic Maltogenic Amylase from
Staphylothermus marinus*
Receivedforpublication,September15,2011,andinrevisedform,January3,2012Published,JBCPapersinPress,January5,2012,DOI10.1074/jbc.M111.304774
Tae-YangJung‡§,DanLi¶1,Jong-TaePark(cid:1),Se-MiYoon§,PhuongLanTran**,Byung-HaOh‡,SˇtefanJanecˇek‡‡§§2,
SungGooPark§,Eui-JeonWoo§¶¶3,andKwan-HwaPark**4
Fromthe‡DepartmentofBiologicalSciences,KAISTInstitutefortheBioCentury,KoreaResearchInstituteofBioscienceand
Biotechnology,Daejeon305-701,Korea,the§MedicalProteomicsResearchCenter,KoreaResearchInstituteofBioscienceand
Biotechnology,Daejeon305-333,Korea,the¶DepartmentofFoodScienceandEngineering,ChangchunUniversity,Changchun
130022,China,the(cid:1)DepartmentofFoodScienceandTechnology,ChungnamNationalUniversity,Daejeon305-764,Korea,the
**DepartmentofFoodServiceManagementandNutrition,SangmyungUniversity,Seoul110-743,Korea,the‡‡Instituteof
MolecularBiology,SlovakAcademyofSciences,SK-84551Bratislava,Slovakia,the§§DepartmentofBiotechnology,Facultyof
NaturalSciences,UniversityofSS.CyrilandMethodius,SK-91701Trnava,Slovakia,andthe¶¶DepartmentofAnalyticalBioscience,
UniversityofScienceandTechnology,Daejeon305-333,Korea
Background:Maltogenicamylasesthatareknowntodateformdimerstoperformhydrolysis.
Results:ThestructureofmaltogenicamylasefromStaphylothermusshowedanoveldomainattheNterminusassociatedwith
theactivesite.
Conclusion:Staphylothermusamylasehasallofitssubstrate-bindingstructuralcomponentsinasinglemonomer.
Significance:Thisisthefirstreportofthenewlyobserveddomainarrangementadoptedbyhyperthermophilicarchaicmalto-
genicamylase.
Staphylothermusmarinusmaltogenicamylase(SMMA)isanovel genicamylasesfromarchaeaandthatdistinguishSMMAfrommod-
extremethermophilemaltogenicamylasewithanoptimaltempera- eratethermophilicormesophilicbacterialenzymes.
tureof100°C,whichhydrolyzes(cid:1)-(1–4)-glycosyllinkagesincyclo-
dextrinsandinlinearmalto-oligosaccharides.Thisenzymehasalong
N-terminalextensionthatisconservedamongarchaichyperthermo- Many organisms live in physically or geochemically extreme
philicamylasesbutisnotfoundinotherhydrolyzingenzymesfrom conditionsthataredetrimentaltomostorganisms.Mosteukary-
the glycoside hydrolase 13 family. The SMMA crystal structure otic organisms cannot tolerate temperatures higher than 50°C,
revealedthattheN-terminalextensionformsanN(cid:1)domainthatis duetothesensitivityofcertaincellularcomponents.Thediscovery
similar to carbohydrate-binding module 48, with the strand-loop- ofhyperthermophilicmicroorganismslivingnear,at,andabove
strandregionformingapartofthesubstratebindingpocketwithsev- 100°Chasrevolutionizedscientificthoughtinthisarea,andmany
eralaromaticresidues,includingPhe-95,Phe-96,andTyr-99.Astruc- scientists have become interested in such systems because they
tural comparison with conventional cyclodextrin-hydrolyzing havedevelopedspecificphysicochemicalcharacteristics.Dueto
enzymes revealed a striking resemblance between the SMMA N(cid:1) these particular properties, enzymes from extremophiles offer
domain position and the dimeric N domain position in bacterial greatpotentialforbasicresearchandforbiotechnologicalapplica-
enzymes.Thisresultsuggeststhatextremophilicarchaeathatliveat tions.Forexample,mostindustrialstarchprocessesrequirehigh
hightemperaturesmayhaveadoptedanoveldomainarrangement temperatures for liquefaction and saccharification. Therefore,
thatcombinesallofthesubstratebindingcomponentswithinamono- there is enthusiastic interest in finding new sources of thermo-
mericsubunit.TheSMMAstructureprovidesamolecularbasisfor stableamylolyticenzymes.
thefunctionalpropertiesthatareuniquetohyperthermophilemalto- Recently, we reported a novel maltogenic amylase from
Staphylothermusmarinus(1)thatwasisolatedfromgeother-
mal sediments from a “black smoker” on the ocean floor (2).
*ThisresearchwassupportedinpartbytheMarineandExtremeGenome
ResearchCenterprogramfromtheMinistryofLand,Transport,andMari- Maltogenic amylase is an enzyme that is widely used in the
timeAffairs,RepublicofKorea,andtheNationalResearchFoundationof starchindustry.Thisenzymeexhibitsdualactivityfor(cid:1)-D-1,4-
Korea(NRF)fundedbytheMinistryofEducation,Science,andTechnology and (cid:1)-D-1,6-glucosidic bond cleavage, which differs from the
(Grants2009-0083290/2011-0017040).
classic(cid:1)-amylasesintheglycosidehydrolase13(GH13)5family
Theatomiccoordinatesandstructurefactors(code4AEE)havebeendepositedin
theProteinDataBank,ResearchCollaboratoryforStructuralBioinformatics,
RutgersUniversity,NewBrunswick,NJ(http://www.rcsb.org/).
1Supported by National Natural Science Foundation of China Grant 5Theabbreviationsusedare:GH13,glycosidehydrolase13;ADA,N-(2-acetamido)-2-
31000760. iminodiaceticacid;ThMA,maltogenicamylasefromThermussp.strainIM6501;
2SupportedbySlovakGrantAgencyVEGAGrant2/0148/11. NPase,neopullulanasefromB.stearothermophilus;CDase,cyclodextrinasefrom
3To whom correspondence may be addressed. Tel.: [phone]; Fax: alkalophilicBacillussp.I-5;TVAIandTVAII,T.vulgarisamylaseIandII,respectively;
82-42-879-8596;E-mail:[email protected]. SMMA,S.marinusmaltogenicamylase;PFTA,P.furiosusthermostableamylase;
4Towhomcorrespondencemaybeaddressed.Tel.:[phone];Fax:82-2- CD,cyclodextrin;CBM,carbohydrate-bindingmodule;r.m.s.,rootmeansquare;
391-8758;E-mail:[email protected]. aa,aminoacids;AMPK,5(cid:1)-AMP-activatedproteinkinase.
MARCH9,2012•VOLUME287•NUMBER11 JOURNALOFBIOLOGICALCHEMISTRY 7979
This is an Open Access article under the CC BY license.
CrystalStructureofSMMA
(3).SMMAhasanoptimaltemperatureof100°Canda109°C TABLE1
meltingtemperaturewithenzymaticactivityunderacidiccon- Datacollectionandstructure-solutionparameters
ditions(pH3.5–5.0),whichisafavorablepropertyforindustrial Parameters Values
applications(2,4).Theconversionofstarchatahightempera- Crystaltype Native
Unitcellparameters(Å) a(cid:6)65.39,b(cid:6)117.51,c(cid:6)199.04
ture and low pH offers several advantages, including higher
Resolution(Å) 50–2.28
substratesolubility,decreasedviscosity,betterbacterialdecon- Spacegroup P222
1 1 1
tamination,andincreasedreactionrates(4–8). Completeness(%) 85.6(72.9)a
R (%)b 4.8(19.8)
The GH13 family includes (cid:1)-amylases and closely related I/s(cid:4)ym(I) 37.1(4.9)
subfamilies,suchasmaltogenicamylase(EC3.2.1.133),neopul- No.ofrefinedatoms:protein/water 11,565/385
R /R (%)c 18.7/23.7
lulanase(EC3.2.1.135),ThermoactinomycesvulgarisamylaseII r.fmac.tso.rdefvreieationbondlength(Å) 0.012
(TVAII), and cyclomaltodextrinase (CDase; EC 3.2.1.54) (3). r.m.s.deviationbondangle(degrees) 1.116
Maltogenic amylase shares catalytic characteristics with Ramachandranplot(%)
Mostfavoredregion 1293(94.6%)
neopullulanase and CDase, which catalyze the hydrolysis of Additionallyallowedregion 74(5.4%)
cyclodextrins(CDs),pullulan,andacarbose,andwhicharecol- Outlierregion 0(0%)
lectivelyknownasCD-hydrolyzingenzymes(9).Theseenzyme aNumbersinparenthesesarestatisticsfromthehighestresolutionshell.
b R (cid:6)(cid:2)I (cid:3)I (cid:2)/I ,whereI istheobservedindividualreflection,andI
specificitieswereproposedtoestablishtheso-calledneopullu- isstyhmeaveorbasgeovaevgralolbssymmetryoebqsuivalents. avg
lanasesubfamilyofthe(cid:1)-amylasefamilyGH13(10),whichis cRfactor(cid:6)(cid:2)Fo(cid:2)(cid:3)(cid:2)Fc(cid:1)/(cid:2)Fo(cid:2),whereFoandFcaretheobservedandcalculatedstruc-
turefactoramplitudes,respectively.R wascalculatedusing5%ofthedata.
currentlyclassifiedasthesubfamilyGH13_20(11).CD-hydro- free
lyzingenzymespossessacommondomainattheNterminus(N
domain) that is involved in substrate binding through a viously (14). The active fractions in the elution buffer were
domain-swappedhomodimericstructure(12–16).Thenarrow, dialyzedagainst50mMTris-HClbuffer(pH7.5).
deepactivesitegeneratedbytheNdomainfromanothersub- CrystallizationandDataCollection—SMMAcrystallization
unitinthedimerisresponsibleforthesubstratepreference,for trialswereconductedusingthesittingdropmethodat18°C.
example, for small substrates and CDs. SMMA has enzyme Wemixed1.5(cid:2)lofa14mg/mlSMMAsolutionwithanequal
activitysimilartotheCD-hydrolyzingenzymeswithbroadsub- volumeofcrystallizationreservoirsolutioncontaining12%pol-
stratespecificity. yethyleneglycol(PEG)4000,2%isopropylalcohol,0.1MADA,
Interestingly, the primary structure analysis of SMMA pH6.5,and0.1MLi SO .Beforedatacollection,rhombus-type
2 4
revealed that most thermostable maltogenic amylases from crystalswerecryocooledto95Kusingacryoprotectantconsist-
archaea, such as S.marinus (1), Thermofilum pendens Hrk5 ingofmotherliquorsupplementedwith25%glycerol.Thecrys-
(17), Thermoplasma volcanium GSS1 (18), and Pyrococcus taldiffractedtoaresolutionof2.28Å,andthedatawerecol-
furiosus(19),havealongermotifattheN-terminalregionthat lectedwitha1°rotationandatotalof340frames.
is220–250aminoacidslong.However,otherbacterialmalto- Structure Determination and Refinement—Diffraction data
genicamylasesandCD-hydrolyzingenzymeshaveN-terminal wereprocessedandscaledusingHKL2000(22).Thestructure
regionswithonly120–140aminoacidsthatintheCAZy(Car- wasdeterminedusingthemolecularreplacementmethodwith
bohydrate-active Enzyme) server have been classified as the thePhaserCCP4suite(23)andtheneopullulanasefromBacil-
carbohydrate-bindingmodule(CBM)family34,basedonthe lusstearothermophilus(ProteinDataBankentry1J0H)withthe
observation that this domain from the related (cid:1)-amylase I N domain omitted. The resulting model was refined through
(TVAI)inT.vulgarisbindscarbohydrates(20,21). modelrebuildingusingCNS(24).COOT(25)wasusedforster-
Becauseitisthemostthermostablemaltogenicamylaseyet eographicmanualrefinementandmodelbuilding.Thestruc-
reported,westudiedtheSMMAstructurefordetailedinforma- ture was validated with PROCHECK (26). Structure-based
tionaboutitsfunction.Inthisstudy,weshowthethree-dimen- sequence alignments were generated using ClustalW (27).
sionalstructureoftheenzyme,whichrevealsauniquedomain Molecularimages,includingschematicsandstickfigures,were
arrangementintheactivesiteassociatedwiththeN-terminal produced using PyMOL (28). The detailed statistics for data
regionthatdistinguishesthearchaealmaltogenicamylasefrom collectionandrefinementarelistedinTable1.
classicbacterialmaltogenicamylases. MolecularModeling—TheSMMAand(cid:3)-cyclodextrinligand
complex model was constructed by overlaying the SMMA
EXPERIMENTALPROCEDURES
structure onto the ThMA complex structure (Protein Data
Protein Purification—The strain Escherichia coli MC1061 Bankentry1GVI).Thesubstratewasoptimizedmanuallyprior
(F-7, araD139, (cid:2)(ara-leu)7696, galE15, galK16, (cid:2)(lac)X74, toenergyminimizationbyusingthesteepestdescentmethod
rpsL,(Strr), hsdR2, (r (cid:3)m (cid:4)), mcrA, and mcrB1) harboring withan8-Åcut-offfor300iterationsusingInsightII(Accerlys,
k k
pSMMA6xHwasculturedfor20hin3litersofLuria-Bertani SanDiego,CA).
broth.Thecellswerecollectedbycentrifugation(8000(cid:5)g,30 AminoAcidSequenceAlignmentsforGenesandProteins—A
min),resuspendedin300mloflysisbuffer(50mMTris-HCl, BLAST(29)searchonSMMA(GenBankTMaccessionnumber
pH 7.5, 300 mM NaCl, and 10 mM imidazole), and sonicated. ABN69720.1) showed that the encoded protein has 23–28%
Thesupernatantwascollectedbycentrifugation(10,000(cid:5)g,30 identitywithotherGH13proteins,includingcyclomaltodextri-
min,4°C)andheatedat70°Cfor20mintoremovethether- nase from Thermococcus sp. B1001 (30), maltogenic amylase
molabileE.coliproteins.Thecrudeenzymewasfurtherpuri- fromThermussp.IM6501(14),neopullulanasefromB.stearo-
fiedusinganickel-NTASuperflow(cid:2)column,asdescribedpre- thermophilus(31),andT.vulgaris(cid:1)-amylaseII(13).
7980 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER11•MARCH9,2012
CrystalStructureofSMMA
FIGURE1.OverallSMMAstructure.a,aschematicoverviewofanSMMAmonomerthatshowstheconservedN,catalytic,andCdomainsinCD-hydrolyzing
enzymeswithanovelN(cid:1)domain.Themonomeriscoloredinaspectrum;theNterminusisinblue,andtheCterminusisinred.b,theSMMAdimerstructureis
shownwitha2-foldaxisperpendiculartotheplane.c,thedimericinterfacebetweentheN(cid:1)domainsisshownwiththeadjacenthydrophobicandcharged
interactions.d,asurfacediagramofthemonomerwithahypotheticalcyclodextrinmolecule(orange)isgeneratedbysuperpositionwiththebinarycomplex
structure(ProteinDataBankentry1GVI)tohighlighttheactivesite.
Kinetic Parameters and Reaction Product Analysis—The domains, which are observed in most CD-hydrolyzing
copperbicinchoninatemethodwasusedtomeasurethecon- enzymes, and an additional novel N-terminal domain, the N(cid:1)
centration of reducing products to determine activity and domain, which was first observed in this study (Fig. 1a). Ini-
kineticparametersofSMMA(15,32).Forthinlayerchroma- tially, the structure was determined and refined to a 2.28 Å
tography (TLC) analysis, the reaction products were spotted resolutionusingmolecularreplacement,withthecatalyticand
ontoWhatmanK5Fsilicagelplates(Whatmanplc,Maidstone, CdomainsofneopullulanasefromB.stearothermophilus(Pro-
UK)anddevelopedusingisopropylalcohol/ethylacetate/water tein Data Bank entry 1J0H) as the template structure. An
(3:1:1,v/v/v)asthesolventsystem.
attempt at molecular replacement with the entire three-do-
AnalysisofSurfaceTyrandTrpResidues—Proximitytothe
mainregionfailed,whichmayhavebeenduetothesignificantly
endofasecondarystructureelementisdefinedasbeingwithin
altered orientation and geometry of the SMMA N domain
4 amino acids for helix and within 1 amino acid for strand, (r.m.s. deviation of 2.3 Å for 463 C(cid:1)atoms). During manual
wheretheterminiareassignedfromtheProteinDataBank(33).
modelbuildingfortheN-terminalregion,theelectrondensity
Structures used in the analysis are Protein Data Bank entries
of the region showed two vague, separate “blobs,” which
1GVI(ThMA),1J0H(NPase),1JI2(TVAII),and1EA9(CDase).
allowedthedetectionoftheN(aa116–219)andN(cid:1)(aa1–115)
Residues in the exposed surfaces were identified using the
domains of SMMA. The SMMA catalytic domain displays a
Areaimolprogram(34).
conserved((cid:3)/(cid:1))8-barrelfoldwithadistinctloop(aa342–397)
RESULTS protruding from the barrel. Most CD-hydrolyzing enzymes
Overall Structure—The crystal structure revealed that have a protruding loop located near the active site, which is
SMMA comprises four domains: the N, catalytic, and C calledtheBdomainandformsaportionofthesubstratebind-
MARCH9,2012•VOLUME287•NUMBER11 JOURNALOFBIOLOGICALCHEMISTRY 7981
CrystalStructureofSMMA
FIGURE2.SequencealignmentforSMMA,PFTA,ThMA,NPase,TVAII,andCDase.ThesequencesofsixCD-hydrolyzingenzymes,twohyperthermophilic
archaicmaltogenicamylases,andfourconventionalbacterialCD-hydrolyzingenzymeswerealignedusingClustalW(27).ThenovelSMMAN(cid:1)domainis
indicatedwithabluedottedline.Thehelix-loop-helixregionsobservedinthestructureareindicatedwithorangedottedlines.The(cid:3)-7-loop7-(cid:3)-8region
protrudingfromtheN(cid:1)domainisindicatedwithapinkdottedline.ThethreeconservedcatalyticresiduesintheGH13familyareindicatedwithblackasterisks.
Red,blue,andgreenhighlightingindicatestheresiduesthatareconservedin100,75,and50%oftheproteins,respectively.Theaccessionnumbersforthese
sequencesareABN69720.1(SMMA),AAL82063.1(PFTAfromPyrococcusfuriosus),AAC15072.1(ThMAfromThermussp.strainIM6501),AAA22622.1(NPase
fromB.stearothermophilus),BAA02473.1(TVAII),andAAA92925.1(CDasefromalkalophilicBacillussp.I-5).
inggrooveforsubsites(cid:3)2,(cid:3)3,or(cid:3)4.However,SMMAhasa model,whichsuggeststhatitmaybeflexible.SMMAformsa
muchlongerinsertionofaa342–397inthisregion,creatinga homodimerviaaninteractionbetweentheadjacent,novelN(cid:1)
larger domain at the entrance of the groove (Fig. 1b). In this domains, which have a 2-fold axis perpendicular to the arc
groove,Tyr-389,whichisinthemiddleofahelixinaa385–391, shape of the (cid:3)-strands’ interface (Fig. 1c). Each monomer is
forms an entrance gate with Tyr-257. Because SMMA and primarily associated through hydrophobic interactions at the
other CD-hydrolyzing enzymes from archaea have this inser- centeroftheregionofaa5–19(Ile-5and-19fromonemolecule
tion, the protruding region might serve as a signature that is againstIle-9fromtheother).Thisinteractionissupplemented
unique to maltogenic amylases from archaea. Another bysaltbridges(Arg-181/Asp-422andArg-50/Glu-198)atboth
sequence-structuralfeatureisthepresenceofaglycineinthei endsofthestrands,whichyielda2140.7Å2interface(Fig.1d).
(cid:4) 4 position after the catalytic nucleophile Asp-442 (Fig. 2) MostCD-hydrolyzingenzymesformdimerswiththeNdomain
because typical maltogenic amylases (i.e. members of the intertwined.However,theSMMAdimerconfigurationisdif-
neopullulanasesubfamily)haveaglutamateinthatpositionasa ferent from previously reported CD-hydrolyzing enzymes, in
partofthefour-residuesignatureVANE(10). that the dimer is arranged with adjacent monomers and an
ADalisearch(35)usingtheSMMAstructureidentifiedthe interfaceunrelatedtotheactivesites.
neopullulanasefromB.stearothermophilus(29%identity)asits CBM48 Topology of the N(cid:1) Domain—The long N-terminal
closeststructuralhomologue,witha2.6År.m.s.deviation(536 regionofSMMAincludestworepeatedmotifs,theNandN(cid:1)
C(cid:1)atoms). The (cid:1)amylase II (TVAII) from T.vulgaris R-47 domains,witha(cid:3)-sandwichfold(6.2År.m.s.deviationfor12
(30%identity)wasidentifiedasthesecondclosesthomologue C(cid:1)atoms)(Fig.3a).Astructuralhomologysearchforthetrun-
witha2.6År.m.s.deviation(537C(cid:1)atoms).Theloopregions catedN(cid:1)domaingeneratedthe(cid:3)-subunitofthe5(cid:1)-AMP-acti-
comprisingaa236–264and653–689hadhighB-factors(61.3 vatedproteinkinase(AMPK)witha1.9År.m.s.deviationover
average) at the surface, and the electron density map for the 80 residues (Z score, 10.2) using the Dali and 1.71 Å over 80
loop of aa 671–677 in the C domain was too weak to build a residues(Zscore,6.2)usingtheSSMserver(36).TheAMPK(cid:3)
7982 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER11•MARCH9,2012
CrystalStructureofSMMA
FIGURE3.CBM48topologyintheN(cid:1)domain.a,superpositionoftheeightCBM48structuresthatcarrytheloopprotrudingfromtheseventhandeighth
(cid:3)-strandstotheN(cid:1)domaininSMMA.b,schematicdiagramshowingtheantiparallel(cid:3)-sandwichfoldfortheNandN(cid:1)domains.Theloopprotrudingfromthe
N(cid:1)domainiscoloredcyan.c,thepositionsoftheNandN(cid:1)domainsatthesurfaceoftheremainingportionoftheSMMAmolecule.d,aromaticresidueslocated
neartheloopprotrudingfromtheN(cid:1)domain.Thedomainhasbeencoloredinaspectrum;theNterminusisinblue,andtheCterminusisinorange.
subunit,whichisknowntobindglycogen,belongstoCBM48 highlyconservedcatalyticresiduesAsp-442,Glu-471,andAsp-
(37).Currently,theCBM48familyhasmorethan3000entries 536atthebottomofthepocket(Fig.4a).Thisactivesitepocket
intheCAZydatabase,and15entrieshavestructuralinforma- islocatedatthecenterofthe((cid:3)/(cid:1))8domain,iscomposedpartly
tionavailable(38).Despitethelowoverallsequencesimilarity, of the catalytic domain regions (residues 285–310, 397–417,
all of the structures superimposed well onto the SMMA N(cid:1) 471–475, 534–546, and 568–594) and the protruding N(cid:1)
domain and share 6–8 (cid:3)-strands from the (cid:3)-sandwich fold, domainloop,andis14.8Ådeep,18.33Ålong,and9.1Åwide
exceptforthelongextendedloopofaa88–110betweenthe7th (Fig.4b).ThepositionsandorientationsoftheAsp-442nucleo-
and 8th (cid:3)strands in SMMA (Fig. 3b). Eight of 15 structures phileandtheGlu-471acid/baseareinappropriaterangesfor
haveprotrudingloopsthatcorrespondtotheloopofaa88–110 retainingenzymaticactivitybecausethecarboxylategroupof
inSMMA,buttheSMMA(cid:3)-strandloopismuchlonger(8.2Å) Asp-536isheldinpositionbyahydrogenbondtothenitrogen
thantheothercorrespondingloops.TheAMPK(cid:3)subunithasa atoms(N(cid:5)1andN(cid:6)2)inHis-535(Fig.4a).Similarly,theoxygen
corresponding loop with the Leu-146 residue at the tip that atoms (O(cid:6)1 and N(cid:6)2) in the Asp-442 carboxylate group are
interactswithcyclodextrinthroughseveralaromaticresidues hydrogen-bonded to His-336 (N(cid:6)2), Tyr-296, and Arg-440
alongthesurfacesoftheother(cid:3)-strandstoaidinthecarbohy- (NH1),andthesepositionsmayberesponsibleformaintaining
dratestackinginteraction(37).IntheSMMAN(cid:1)domain,Phe- the ionization state of the nucleophile. The pocket contains
95,Phe-96,andTyr-99arealongtheloop,andnumerousaro- several aromatic residues; Phe-404 and Phe-405 form subsite
maticresidues,suchasTyr-43,Phe-46,andPhe-77,lieonthe (cid:3)1, and Tyr-296 provides the essential stacking interaction
(cid:3)-sandwichfoldsurfaces(Fig.3d).Interestingly,theSMMAN(cid:1) withthesugarringatsubsite(cid:3)1thatisconservedinGH13and
domainisinthepositionoftheNdomaininotherneopullula- formsahydrogenbondwiththenucleophileAsp-442.
nasesubunits,withitsprotrudingloopbeingextendedtothe TheactivesitepocketsinbacterialCD-hydrolyzingenzymes
active site of the catalytic domain. With this orientation and aregeneratedbytheNdomainfromtheothersubunitandyield
geometry,theSMMAN(cid:1)domainformspartoftheactivesite agroovethatisslightlyextendedbetweenthecatalyticdomain
pocketviaresiduesPhe-95,Phe-96,andTyr-99,whichmakeup andtheNdomain,probablyabovesubsites(cid:4)3and(cid:4)4(Fig.4d).
thesubstratebindinggroove. Incomparison,theSMMAactivesitepocketisgeneratedbythe
ShapingActiveSitewithNovelN(cid:1)Domain—InSMMA,the N(cid:1) domain of the same subunit without a significant groove
substrate binding pocket was easily identified by the three above the subsites (cid:4)3 and (cid:4)4, at which the position of the
MARCH9,2012•VOLUME287•NUMBER11 JOURNALOFBIOLOGICALCHEMISTRY 7983
CrystalStructureofSMMA
FIGURE4.SMMAactivesite.a,catalytic(red)andrelatedsubsiteresidues(cyan)areshownassticks.Thehydrogenbondnetworkintheactivesiteisshownwith
theadditionalaromaticN(cid:1)domainresidues(blue).b,asurfacerepresentationoftheactivesitepocketisshownwithahypotheticalCDmoleculemodeledat
theactivesite.c,astructuralcomparisonoftheactivesitesinSMMA(left)andNPase(right)fromB.stearothermophilus(ProteinDataBankentry1J0H);the
substrate-bindinggroovesattheactivesitesareshown.
putativesubsite(cid:4)3isfilledandblockedbyPhe-405andPhe-77 SMMAThermostability—ThereportedSMMAmeltingtem-
from the N(cid:1) domain (Fig. 4c). In addition, the N domain in peratureis109°C;thus,itisthemostthermostablemaltogenic
bacterialCD-hydrolyzingenzymesprotrudesabovethesurface amylasereportedtodate.Previously,thehighfrequencyofsur-
andformsastructurallidfortheactivesitepocket,whereasthe facehydrophobicresidueswithbulkysidechains,suchasTrp
SMMAN(cid:1)domaindoesnothavethestructurallid,whichmay andTyrinthe(cid:1)-helicesand(cid:3)-strandtermini,werereportedto
explainthelackofSMMAtransferactivity.Glu-332,whichis berelatedtothethermostabilityofmanyproteins(33).Thus,
knowntobeakeyresidueinThMAtransglycosylationactivity, wehaveinvestigatedthedistributionofTrpandTyrincom-
isreplacedbyGlyinSMMA(39).Becausetheactivesitepocket parison with CDase, ThMa, neopullulanase, and TVAII. The
issignificantlyalteredinSMMA,wehaveexaminedthedegra- structuralanalysisshowedthatasignificantlyhighproportion
dation pattern with the long-chain substrates amylose and of Tyr and Trp residues are located on the solvent-accessible
amylopectin.AsshowninFig.5,SMMAhydrolyzesbothamy- surfacethroughouttheentiremolecule(Table3).Althoughthe
lose and amylopectin to produce maltose and glucose exclu- totalnumberofTyrandTrpresiduesisnotsignificantlyhigher
sively. A kinetic analysis in Table 2 showed that SMMA has thanintheotherenzymes,67(9.6%)comparedwith44(7.7%),
lowerK valueforamylosesubstrate(0.27)thanforamylopec- theseresiduesareprimarilylocatedonthesurfaceinSMMA.
m
tin(0.51),whichexhibitsasimilarpatterntoclassicmaltogenic Fig.6showsthattherelativesurfacedistributionofTyrandTrp
amylases.However,K valuesforbothsubstratesweresignifi- attheterminiofthesecondarystructureismuchwiderthanin
m
cantlylowerthanthoseofsomemaltogenicenzymes.Foramy- those bacterial enzymes. Many extremophiles use sugars as a
losesubstrate,itis5-foldlowerthanthatofCDase(0.27versus compatiblesolutetoeaseosmoticstress,andsuchsolutessta-
1.51)and70-foldlowerthanthatofThMA(0.27versus19.7). bilizeproteins,whichmayexplaintheabundanceofaromatic
Foramylopectin,itisatleast2ordersofmagnitudelowerthan residuesthatcanbindcarbohydratesugarsattheproteinsur-
CDase and ThMA (0.51 versus 55.15 and 0.51 versus 44.5, face(33,40–42).AminoacidanalysisshowedthatSMMAhas
respectively),whereasthespecificityconstantk /K issimilar asignificantlyhigherfrequencyofIle(74,10.6%)thandothe
cat m
(1.17versus0.92and1.17versus3.03,respectively),whichsug- otherfourenzymes(average24,4.0%),butithassignificantly
gests that SMMA has a high affinity for polysaccharide sub- lower Gln content (9, 1.3%). Ile occurs often in thermophilic
strates.SMMAshowedauniquelyhigherk valuefor(cid:7)-CD proteins,andGlnisalabileaminoacidthatisdeaminatedat
cat
substratethanfor(cid:1)-CDand(cid:3)-CD. hightemperatures,whichsuggeststhatknownstructuralfac-
7984 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER11•MARCH9,2012
CrystalStructureofSMMA
evolution of specificities, rather than the evolution of species
(61), the detailed function for CBM48 is unknown. The only
exceptionisrepresentedbythe(cid:3)-subunitofAMPK(37),which
showedthatCBM48isaseparatedomainthatbindscyclodex-
trin to increase its binding ability for the glucosyl polymeric
structures commonly found in glycogen (62). Despite the
wealthofstructuralinformationonCBM48(36,52,55,63–67)
(for a review, see Ref. 68)), this paper is, to the best of our
knowledge,thefirsttodemonstratethatanindependentCBM
domainfoldstointeractwiththecatalyticdomainandpartici-
patesinsubstratebindingattheactivesite.Whileitisadjacent
tothecatalyticdomain,CBM48intheN(cid:1)domaincontactsthe
active site with the long extended loop that was previously
shown to interact with cyclodextrin molecules in the AMPK
(cid:3)-subunit(37).TheSMMAN(cid:1)domainextendedloophassev-
eral aromatic residues, which is a suitable architecture for a
stacking interaction with carbohydrate sugar rings. The
reducedK valuestowardamyloseandamylopectinreported
m
inthisstudymayreflectthecharacteristicbindingofCBMto
carbohydrates. For CBM48 function at the active site, it is
related to substrate specificity in the glycogen debranching
enzymes from Deinococcus geothermalis and Deinococcus
radiodurans(69).TheCBM48determinesthebranchingpat-
ternofglycogenattheactivesite,whichsuggestsafunctional
rolesimilartotheSMMAN(cid:1)domain.Thus,itwouldbeinter-
estingtorelatetheproteinstructuretoitsfunction(69).
FIGURE5.SMMAenzymaticcharacteristicsforamyloseandamylopectin
substrates.AtimecourseanalysisofSMMAwithamyloseandamylopectin Previously,wereportedthatsubstratebindinginThMAwas
frompotatowasshown.Thereactionproductsofglucoseandmaltosewere influenced by the direction and arrangement of several loops
furtheranalyzed(reactionconditions:SMMA0.6unit/mg ,substrate0.25%,
sub located in the N domain (15). The striking similarity of the
50mMNaOAc,pH5.0,92°C). position and orientation of the SMMA N(cid:1) domain to the N
torsinvolvedinthermostabilitycorrelatewellwiththeSMMA domaininbacterialmaltogenicamylasesinthisstudyindicates
structure(43–45). acommonfunctionalroleinthesubstratebindingattheactive
site(Fig.7).Interestingly,theSMMANdomainlacksthecor-
DISCUSSION
responding functional residues, such as Tyr-45 and Trp-47,
Certain CD-hydrolyzing amylases have much longer whichareinvolvedinsubstraterecognitioninThMA(15).We
extendedregionsintheNterminus,despitetheirhighsequence suggestthattheSMMAN(cid:1)domainplaysthefunctionalroleof
homologywiththeremainingclassicCD-hydrolyzingenzymes. the N domain in moderate thermophile or mesophile malto-
Thus, the functional role of this region in amylase activity is genicamylases.
especially interesting. Given the high sequence homology of TheinterfacebetweentheN(cid:1)domainandotherdomainsin
thisextendedregionamongarchaicenzymes,itislikelythatthe the SMMA monomeric subunit showed much stronger and
CD-hydrolyzing enzymes from hyperthermophilic archaea, tighterinteractionsthandidtheinterfaceoftheNdomainin
such as Staphylothermus hellenicus (46), Thermococcus baro- bacterial maltogenic amylases. The N(cid:1) domain is primarily
philus (47), Thermococcus kodakarensis (48), Thermococcus associated with a 1946.0 Å2 buried interface via hydrophobic
onnurineus(49),T.pendens(50),T.volcaniumGSS1(18),and interactions,whereastheNdomaininThMAhasadistribution
P.furiosus(51),haveextradomainswiththeCBM48fold. of highly charged residues at the interface with a 1338.8-Å2
CBMs have been divided into 64 families based on amino buried surface. It has been reported that high salt concentra-
acidsequencesimilarity(38).Previously,CBMswerethought tionschangetheoligomericstateofbacterialmaltogenicamy-
tobemotifsthatarefunctionallyindependentfromthecata- lases(70).Althougholigomerizationmightnotbenecessaryfor
lyticdomainslocateddistaltotheactivesites,andithasbeen theactivityoftheGH13CD-hydrolyzingenzymes,itdoescon-
proposedthattheyenhancetheinteractionbetweenthecarbo- tributetotheirhighspecificity(71).Thechargedgroupinthe
hydrate substrate and protein (52–55). For many amylolytic hydrophobicinterfacemayhavedirecteffectsonthermalresil-
enzymes,CBMswerereportedlyinvolvedinstarchhydrolysis ienceandserveasadoorwayforwatermolecules.Hightemper-
bydisruptingthestarchgranulestructure,whichallowedfora atureswouldcontributetothermaldestabilizationbyintroduc-
concentrationofcatalyticdomainsonthesurfaceandcarbohy- ing water permeation, which would result in structure
drate starch hydrolysis by proximity (56–59). For example, perturbation. Taken together, a modified structure that posi-
CBM20inCGTasesisinvolvedinbindingandguidinglinear tionsallofthecomponentsforsubstraterecognitionwithina
starchchains(60).AlthoughtheevolutionaryhistoryofCBM48 monomermayhavebeenadoptedtoallowthearchaeahyper-
has been thoroughly elucidated, indicating that it reflects the thermophilic maltogenic enzymes to retain activity at high
MARCH9,2012•VOLUME287•NUMBER11 JOURNALOFBIOLOGICALCHEMISTRY 7985
CrystalStructureofSMMA
TABLE2
KineticparametersforsomesubstratesofSMMA
Enzyme Substrate k K k /K Source/Reference
cat M cat M
s(cid:3)1 mgml(cid:3)1 mM s(cid:3)1mlmg(cid:3)1 s(cid:3)1mM(cid:3)1
SMMA Amylose 0.80 0.27 2.90 Thisstudy
Amylopectin 0.59 0.51 1.17 Thisstudy
(cid:1)-Cyclodextrin 1.45 0.43 3.37 Thisstudy
(cid:3)-Cyclodextrin 5.36 1.48 3.62 Thisstudy
(cid:7)-Cyclodextrin 29.10 2.68 10.86 Thisstudy
CDaseI-5 Amylose 22.11 1.52 14.60 Ref.5
Amylopectin 50.36 55.15 0.92 Ref.5
(cid:3)-Cyclodextrin 78.18 0.515 151.67 Ref.5
ThMA Amylose 96.4 19.7 4.89 Ref.72
Amylopectin 135 44.5 3.03 Ref.72
(cid:3)-Cyclodextrin 126 0.174 726.0 Ref.15
TVAII (cid:3)-Cyclodextrin 43.40 1.31 33.13 Ref.16
TABLE3
RelationshipbetweenthermostabilityandtheratioofresiduesTrpandTyr
Totalnumber Totalnumber Surfacenumber/
Enzyme Temperaturea ofresidues (Tyr/Trp)b Terminalnumberc Surfacenumberd totalnumber Relativenumbere
ºC %
SMMA 100 696 67 27 20 2.87 100
ThMA 60 588 45 18 4 0.68 23.7
NPase 50 588 48 15 9 1.53 53.3
TVAII 50 585 39 13 4 0.68 23.7
CDase 40–50 583 42 18 4 0.68 23.7
aThetemperatureindicatestheoptimaltemperaturesformaximumenzymeactivity.
bThenumberoftotalTyrandTrpresidues.
cThenumberofTyrandTrpresiduesneartheendofasecondarystructureelement.
dThenumberofTyrandTrpresiduesneartheendofasecondarystructureelementandexposedatthesurface.
eTherelativevalueofthesurfacenumbertothesurfacenumberofSMMA.
FIGURE6.SurfacedistributionofaromaticresiduesTyrandTrpinCD-hydrolyzingenzymes.AromaticringsontheproteinsurfacearecoloredredinSMMA
(a),ThMA(b),NPas(c),andCDase(d).Residuesattheterminiof(cid:3)-strandsand(cid:1)-helicesarehighlightedbycyanandmagenta,respectively.ThenumberofTyr
andTrpresiduesthatarelocatedattheterminiofsecondarystructureelementsandexposedatthesurfaceisindicated.
7986 JOURNALOFBIOLOGICALCHEMISTRY VOLUME287•NUMBER11•MARCH9,2012
CrystalStructureofSMMA
FIGURE7.SchematicviewoftheSMMAactivesiteconfiguration.DomainsinvolvedinthesubstratebindingaredrawnschematicallyforSMMA(left)andfor
bacterialCD-hydrolyzingenzymes(right)toshowallofthecomponentsformaltogenicactivityinasinglemonomer.
temperatures.ThestructuralfeaturesofSMMAmayalsopro- Towardsimprovedfunctionalannotationsof(cid:1)-amylase-relatedproteins.
videamolecularbasisforengineeringsubstratepreferenceand ProteinEng.Des.Sel.19,555–562
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Acknowledgment—We thank the beamline staff at Photon Factory,
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