Preparedbycontrolledhydrolysisofcarobgalactomannanwithβ-mannanaseandα-galactosidase.Man:Gal=98:1(DP~15).Borohydridereducedtoremovebackgroundcolour.Anexcellentsubstrateforendo-1,4-β-D-mannanase.
CompletegenomeofanewFirmicutesspeciesbelongingtothedominanthumancolonicmicrobiota(‘Ruminococcusbicirculans’)revealstwochromosomesandaselectivecapacitytoutilizeplantglucans.
Wegmann,U.,Louis,P.,Goesmann,A.,Henrissat,B.,Duncan,S.H.&Flint,H.J.(2014).
EnvironmentalMicroBIOLOGy,16(9),2879–2890.
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Therecentlyisolatedbacterialstrain80/3representsoneofthemostabundant16SrRNAphylotypesdetectedinthehealthyhumanlargeintestineandbelongstothe
RuminococcaceaefamilyofFirmicutes.Thecompletedgenomesequencereportedhereisthefirstforamemberofthisimportantfamilyofbacteriafromthehumancolon.Thegenomecomprisestwolargechromosomesof2.24and0.73Mbp,le
ADIngustoproposethename
Ruminococcusbicirculansforthisnewspecies.Analysisofthecarbohydrateactiveenzymecomplementsuggestsan
ABIlitytoutilizecertainhemicelluloses,especiallyβ-glucansandxyloglucan,forgrowththatwasconfirmedexperimentally.Theenzymaticmachineryenablingthedegradationofcelluloseandxylanbyrelatedcellulolytic
ruminococciishoweverlackinginthisspecies.Whilethegenomeindicatedthecapacitytosynthesizepurines,pyrimidinesandall20aminoacids,onlygenesforthesynthesisofnicotinate,NAD
+,NADP
+andcoenzymeAweredetectedamongtheessentialvitaminsandco-factors,resultinginmultiplegrowthrequirements.
Invivo,thesegrowthfactorsmustbesuppliedfromthediet,hostorothergutmicroorganisms.OtherfeaturesofecologicalinterestincludetwotypeIVpilins,multipleextracytoplasmicfunction-sigmafactors,aureaseandabilesalthydrolase.
Roleofglycosidephosphorylasesinmannoseforagingbyhumangutbacteria.
Ladevèze,S.,Tarquis,L.,Cecchini,D.A.,Bercovici,J.,André,I.,Topham,C.M.,Morel,S.,Laville,E.,Monsan,P.,Lombard,V.,Henrissat,B.&Potocki-Véronèse,G.(2013).JournalofBiologicalChemistry,288(45),32370-32383.
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Tometabolizebothdietaryfiberconstituentcarbohydratesandhostglycansliningtheintestinalepithelium,gutbacteriaproduceawiderangeofcarbohydrate-activeenzymes,ofwhichglycosidehydrolasesarethemaincomponents.Inthisstudy,wedescribetheabilityofphosphorylasestoparticipateinthebreakdownofhuman
N-glycans,fromananalysisofthesubstratespecificityofUhgbMP,amannosidephosphorylaseoftheGH130proteinfamilydiscoveredbyfunctionalmetagenomics.UhgbMPisfoundtophosphorolyzeβ-D-Manp-1,4-β-D-Glc
pNAc-1,4-D-Glc
pNAcandisalsoahighlyefficientenzymetocatalyzethesynthesisofthisprecious
N-glycancoreoligosaccharidebyreversephosphorolysis.AnalysisofsequenceconservationwithinfamilyGH130,mappedonathree-dimensionalmodelofUhgbMPandsupportedbysite-directedmutagenesisresults,revealedtwoGH130subfamiliesandallowedtheidentificationofkeyresiduesrespons
IBLeforcatalysisandsubstratespecificity.Theanalysisofthegenomiccontextof65knownGH130sequencesbelongingtohumangutbacteriaindicatesthattheenzymesoftheGH130_1subfamilywouldbeinvolvedinmannancatabolism,whereastheenzymesbelongingtotheGH130_2subfamilywouldratherworkinsynergywithglycosidehydrolasesoftheGH92andGH18familiesinthebreakdownof
N-glycans.TheuseofGH130inhibitorsastherapeuticagentsorfunctionalfoodscouldthusbeconsideredasaninnovativestrategytoinhibit
N-glycandegradation,withtheultimategoalofprotecting,orrestoring,theepithelialbarrier.
Arevisedarchitectureofprimarycellwallsbasedonbiomechanicalchangesinducedbysubstrate-specificendoglucanases.
Park,Y.B.&Cosgrove,D.J.(2012).PlantPhysiology,158(4),1933-1943.
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Xyloglucaniswidelybelievedtofunctionasatetherbetweencellulosemicrofibrilsintheprimarycellwall,limitingcellenlargementbyrestrictingtheabilityofmicrofibrilstoseparatelaterally.Totestthebiomechanicalpredictionsofthis“tetherednetwork”model,weassessedtheabilityofcucumber(Cucumissativus)hypocotylwallstoundergocreep(long-term,irreversibleextension)inresponsetothreefamily-12endo-β-1,4-glucanasesthatcanspecificallyhydrolyzexyloglucan,cellulose,orboth.Xyloglucan-specificendoglucanase(XEGfromAspergillusaculeatus)failedtoinducecellwallcreep,whereasanendoglucanasethathydrolyzesbothxyloglucanandcellulose(Cel12AfromHypocreajecorina)inducedahighcreeprate.Acellulose-specificendoglucanase(CEGfromAspergillusniger)didnotcausecellwallcreep,eitherbyitselforincombinationwithXEG.Testswithadditionalenzymes,includingafamily-5endoglucanase,confirmedtheconclusionthattocausecreep,endoglucanasesmustcutbothxyloglucanandcellulose.Similarresultswereobtainedwithmeasurementsofelasticandplasticcompliance.BothXEGandCel12Ahydrolyzedxyloglucaninintactwalls,butCel12AcouldhydrolyzeaminorxyloglucancompartmentrecalcitranttoXEGdigestion.XyloglucaninvolvementintheseenzymeresponseswasconfirmedbyexperimentswithArabidopsis(Arabidopsisthaliana)hypocotyls,whereCel12Ainducedcreepinwild-typebutnotinxyloglucan-deficient(xxt1/xxt2)walls.Ourresultsareincompatiblewiththecommondepictionofxyloglucanasaload-bearingtetherspanningthe20-to40-nmspacingbetweencellulosemicrofibrils,buttheydoimplicateaminorxyloglucancomponentinwallmechanics.Thestructurallyimportantxyloglucanmaybelocatedinlimitedregionsoftightcontactbetweenmicrofibrils.
Mannansandendo-β-mannanases(MAN)inBrachypodiumdistachyon:expressionprofilingandpossibleroleoftheBDMANgenesduringcoleorhiza-limitedseedgermination.
González-Calle,V.,Barrero-Sicilia,C.,Carbonero,P.&Iglesias-Fernández,R.(2015).JournalofExperimentalBotany,66(13),3753-3764.
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ImmunolocalizationofmannansintheseedsofBrachypodiumdistachyonrevealsthepresenceofthesepolysaccharidesintherootembryoandinthecoleorhizaintheearlystagesofgermination(12h),decreasingthereaftertothepointofbeinghardlydetectedat27h.Concurrently,theactivityofendo-β-mannanases(MANs;EC3.2.1.78)thatcatalysethehydrolysisofβ-1,4bondsinmannanpolymers,increasesasgerminationprogresses.The MAN genefamilyisrepresentedbysixmembersintheBrachypodiumgenome,andtheirexpressionhasbeenexploredindifferentorgansandespeciallyingerminatingseeds.TranscriptsofBdMAN2,BdMAN4andBdMAN6accumulateinembryos,withamaximumat24–30h,andaredetectedinthecoleorhizaandintherootbyinsituhybridizationanalyses,beforerootprotrusion(germinationsensustricto).BdMAN4isnotonlypresentintheembryorootandcoleorhiza,butisabundantinthede-embryonated(endosperm)imbibedseeds,whileBdMAN2andBdMAN6arefaintlyexpressedinendospermduringpost-germination(36–42h).BdMAN4andBdMAN6transcriptsaredetectedinthealeuronelayer.ThesedataindicatethatBdMAN2,BdMAN4andBdMAN6areimportantforgerminationsensustrictoandthatBdMAN4andBdMAN6mayalsoinfluencereservemobilization.Whetherthecoleorhizainmonocotsandthemicropylarendospermineudicotshavesimilarfunctions,isdiscussed.
PurificationandCharacterizationofaThermostableβ-mannanasefromBacillussubtilisBE-91:PotentialApplicationinInflammatoryDiseases.
Cheng,L.,Duan,S.,Feng,X.,Zheng,K.,Yang,Q.&Liu,Z.(2016).BioMedResearchInternational,ArticleID6380147.
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β-mannanasehasshowncompellingbiologicalfunctionsbecauseofitsregulatoryrolesinmetabolism,inflammation,andoxidation.Thisstudyseparatedandpurifiedtheβ-mannanasefromBacillussubtilisBE-91,whichisapowerfulhemicellulose-degradingbacteriumusinga“two-step”methodcomprisingultrafiltrationandgelchromatography.Thepurifiedβ-mannanase(about28.2 kDa)showedhighspecificactivity(79,859.2 IU/mg).TheoptimumtemperatureandpHwere65°Cand6.0,respectively.Moreover,theenzymewashighlystableattemperaturesupto70°CandpH4.5-7.0.Theβ-mannanaseactivitywassignificantlyenhancedinthepresenceofMn+,Cu2+,Zn2+,Ca2+,Mg2+,andAl3+andstronglyinhibitedbyBa2+,andPb2+.KmandVmaxvaluesforlocustbeangumwere7.14 mg/mLand107.5 μmol/min/mLversus1.749 mg/mLand33.45 µmol/min/mLforKonjacglucomannan,respectively.Therefore,β-mannanasepurifiedbythisworkshowsstabilityathightemperaturesandinweaklyacidicorneutralenvironments.Basedonsuchdata,theβ-mannanasewillhavepotentialapplicationsasadietarysupplementintreatmentofinflammatoryprocesses.
Aflexibleloopformannanrecognitionandactivityenhancementinabifunctionalglycosidehydrolasefamily5.
Liang,P.H.,Lin,W.L.,Hsieh,H.Y.,Lin,T.Y.,Chen,C.H.,Tewary,S.K.,Lee,H.L.,Yuan,S.F.,Yang,B.,Yao,J.Y.&Ho,M.C.(2017).BiochimicaetBiophysicaActa(BBA)-GeneralSubjects,InPress.
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Background:Anarrayofglycosidehydrolaseswithmultiplesubstratespecificitiesarerequiredtodigestplantcellwallpolysaccharides.Cel5EfromClostridiumthermocellumandCel5AfromThermotogamaritimaaretwoglycosidehydrolasefamily5(GH5)enzymeswithhighsequenceandstructuralsimilarity,butnotablypossessdifferentsubstratespecificities;theformerisabifunctionalcellulase/xylanaseandthelatterisacellulase/mannanase.AspecificloopinTmCel5A,Tmloop,isoneofthemoststructurallydivergentregionscomparedtoCtCel5Eandinteractswithsubstrates,suggestingtheimportanceformannanrecognition.Method:ATmloopinsertedCtCel5EanditsrelatedmutantswereproducedtoinvestigatetheroleofTmloopincatalysis.CrystalstructureofCtCel5E-TmloopF267Afollowedbysite-directmutagenesisrevealsthemechanism.RtCelB,ahomologwithTmloopwasidentifiedtohavemannanaseactivity.Result:TmloopincorporationenablesCtCel5Etogainmannanaseactivity.Tyr270,His277,andTrp282intheTmloopareindispensableforCtCel5ETmloopcatalysis,andweakeninghydrophobicenvironmentneartheTmloopenhancesenzymekcat.UsingournewlyidentifiedloopmotiftosearchforstructurallyconservedhomologsinothersubfamiliesofGH5,weidentifiedRtCelB.Thishomolog,originallyannotatedasacellulasealsopossessesmannanaseandxylanaseactivities.Conclusion:OurstudiesshowthatTmloopenhancesGH5enzymepromiscuityandplaysaroleincatalysis.Generalsignificance:ThestudyidentifiedaloopofGH5formannanrecognitionandcatalysis.Weakeningthehydrophobicenvironmentneartheloopcanalsoenhancetheenzymecatalyticrate.OurfindingsprovideanewinsightonmannanrecognitionandactivityenhancementofGH5.