Polysaccharide degradation by lytic polysaccharide monooxygenases

(1)

Polysaccharide degradation by lytic polysaccharide monooxygenases

Zarah Forsberg

¹

, Morten Sørlie

¹

, Dejan Petrovi c

¹

,

Gaston Courtade

²

, Finn L Aachmann

²

, Gustav Vaaje-Kolstad

¹

, Bastien Bissaro

¹

, A˚smund K Røhr

¹

and Vincent GH Eijsink

¹

Thediscoveryofoxidativecleavageofglycosidicbondsby enzymescurrentlyknownaslyticpolysaccharide

monooxygenases(LPMOs)hashadamajorimpactonour currentunderstandingoftheenzymaticconversionof recalcitrantpolysaccharidessuchaschitinandcellulose.The numberofLPMOsequencefamilieskeepsexpandingand novelsubstratespecificitiesandbiologicalfunctionalitiesare beingdiscovered.ThecatalyticmechanismoftheseLPMOs remainssomewhatenigmatic.Recently,novelinsightshave beenobtainedfromstudiesofenzyme–substratecomplexes byX-raycrystallography,EPR,NMR,andmodeling.

Furthermore,ithasbeenshownthatLPMOsmaycarryout peroxygenasereactions,atmuchhigherratesthan monooxygenasereactions,whichaffectsourunderstanding andexploitationofthesepowerfulenzymes.

Addresses

1FacultyofChemistry,BiotechnologyandFoodScience,Norwegian UniversityofLifeSciences(NMBU),N-1432A˚s,Norway

2NOBIPOL,DepartmentofBiotechnologyandFoodScience, NorwegianUniversityofScienceandTechnology(NTNU),SemSælands vei6/8,N-7491Trondheim,Norway

Correspondingauthor:Eijsink,VincentGH(vincent.eijsink@nmbu.no)

CurrentOpinioninStructuralBiology2019,59:54–64

ThisreviewcomesfromathemedissueonCatalysisandregulation EditedbyPhilAColeandAndreaMattevi

ForacompleteoverviewseetheIssueandtheEditorial Availableonline1stApril2019

https://doi.org/10.1016/j.sbi.2019.02.015

0959-440X/ã2019TheAuthors.PublishedbyElsevierLtd.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creative- commons.org/licenses/by-nc-nd/4.0/).

Introduction

The role of redox enzymes in biomass conversion is gaininginterest,assuchenzymesmaypromotethecon- version of recalcitrant polysaccharides [1,2,3,4,5]. In comparison to canonical glycoside hydrolases, the role of redoxenzymes(potentially) actingonplant cellwall polysaccharideshasremainedunclear.Forexample,fun- galcellobiosedehydrogenase(CDH)hasbeenstudiedfor decades[6],withoutfindingaclearroleforthisenzyme,

although multiple biological roles have been proposed [7]. A major breakthrough came in 2010 when Vaaje- Kolstadet al. described oxidative cleavageof glycosidic bondsbyenzymesknowntodayasLyticPolysaccharide MonoOxygenases (LPMOs). LPMOs are mono-copper enzymes that, in the presence of an external electron donor,catalyzehydroxylationofoneofthecarbons(C1or C4)inthescissileglycosidicbond,whicheventuallyleads to bond breakage by an elimination reaction[3,8,9]. In contrasttohydrolyticenzymes,whichinteractwithsingle polysaccharidechains,LPMOscanactonpolysaccharide chainsthatresideinacrystallineenvironment.Thisleads todisruptionofthestructure,makingthecellulosemore accessibleforhydrolyticenzymes[10–12].

Since their discovery in 2010, LPMOs have been intenselystudied,duetotheirgreatscientificandindus- trialinterest.LPMOsareabundantinnature,inparticular in fungi [13], and they catalyze a powerful oxidation reactionthatinvolvesmultiplefactorsthatmaybehard to control. Majordevelopmentsof recentyears include the discovery of novel LPMO families [14,15] and increased insight into enzyme–substrate interactions from X-ray and neutron crystallographic, EPR, NMR and modelingstudies [16,17,18,19,20–22,23]. Fur- thermore,ithasbeendiscoveredthatnextto,orperhaps evenratherthan,carryingoutamonooxygenasereaction (R-H+O2+2e+2H⁺!R-OH+H2O), LPMOs carry out peroxygenation of their substrate (R-H+H2O2! R-OH+H2O) [24]. Both reaction mechanisms have been intensely studied using computational methods [25,26,27,28] and kinetics [29,30]. In a comprehen- sivereviewonoxidoreductases(potentially) involvedin lignocellulose conversion, Bissaro et al. have discussed theserecentdiscoveriesinbothabiologicalandapplied perspective[4].

AllLPMOsdescribedsofarhaveasimilaroverallthree- dimensionalstructureandaconserved,highlycharacter- isticsurface-locatedcatalyticmono-coppersite [31].As todate, thesequence-based classificationsystemof the CAZydatabase[32]placesLPMOsintosixfamiliesofso- called ‘auxiliary activities’ (AA9-11 and AA13-15). The large sequence diversity and observed differences in substrate specificity (Figure 1), as wellas the fact that LPMOsoccurinseveralcladesofthetreeoflife,indicate that LPMOs may be involved in biological processes

(2)

Figure1

CBM1 CBM2 CBM3 CBM5 CBM20 CBM73

AA10

AA13

(24.8 kDa, 227 AA)

AA15

AA11

AA14

X278 GH5 GH18 UKD

Mixed C1/C4 Cellulose C1 Cellulose

C1 Cellulose C4 Cellulose

C1 Xylan C1 Chitin

C1 Chitin & cellulose C1 Starch

Xyloglucan, mixed-linkage glucan, glucomannan

Xylan, xyloglucan & mixed-linkage glucan (** mixed C1/C4 activity)

Cello-oligosaccharides, mixed-linkage glucan, xyloglucan, glucomannan, xylan and xylohexaose (** mixed C1/C4 activity)

(19.7 kDa, 178 AA)

(22.4 kDa, 211 AA)

(29.4 kDa, 262 AA) (21.4 kDa, 193

AA)

9

5

5 8

8

8 1

1 2

2

2 4

4 6

6

7

3

3 Mixed C1/C4

Cellulose

& C1 Chitin

(24.5 kDa, 221 AA)

AA9

Tree scale: 1

Current Opinion in Structural Biology

PhylogenetictreeofLPMOs.

Thetreewasbuiltfrom68sequences,whichrepresentthelargemajorityoffunctionallycharacterizedLPMOs.Theunderlyingsequencealignment wasbasedoncatalyticdomainsonlyandonstructuralinformationfromthreeselectedLPMOs(TaAA9A,PDB:2YET;SmAA10A,PDB:2BEM;

AoAA13,PDB4OPB;underlinedinthefigure).The68sequenceswerealignedusingtheT-CoffeeExpressoonlinetool.TheresultingMSAwas employedasinputtobuildthefinalphylogenetictreeusingPhyMLavailableviatheonlineplatformPhylogeny.fr.ThenamesofLPMOswitha knownthree-dimensionalstructureareprintedinboldface.Theoccurrenceofadditionaldomains,forexample,CBMs,GHs,andunknown domains(UKD)isindicatedbysymbols.Thedominatingsubstratespecificityandoxidativeregioselectivity(C1,C4,ormixedC1/C4)foreach

(3)

other than biomass degradation,such as viral virulence [33] and bacterial pathogenicity [34,35]. It has been suggestedthat therecentlydiscoveredAA15s, found in arthropods,algae,oomycetesandcomplexanimals,playa role in development and food digestion [14]. Itseems likely that LPMOs have multiple biological roles that remaintobediscovered.

Theinteraction ofLPMOs with substrates One of the major challenges in understanding LPMO catalysis lies in the insoluble nature of their substrates andtheanalyticalproblemsthisentails.SomeAA9LPMOs (i.e. Group 3 in Figure 1) act on shorter soluble cello- oligosaccharides[36]and, whilethebiological relevance of thisactivity may bequestioned, theseLPMOsmake goodcandidatesforco-crystallizationandsoakingtrialsto generateenzyme–substratecomplexes.In2016,Frandsen etal.were thefirsttoshowacrystalstructureofanAA9 LPMO in complex with cello-oligosaccharide ligands [18].Nexttoshowingthatligand-bindingisdominated bypolarinteractions(seealsoCourtadeetal.[17]),thedata providedinsightintohowsubstrate-bindingaffectsactive sitegeometry,includingthecoppercoordinationsphere.

In the enzyme–substrate complex (Figure 2a), His1 stacks with the +1 sugar, and the space where the non-reduced LPMO (i.e. LPMO-Cu(II)) would bind an axial water (black star in Figure 2a) is filled by the C6-hydroxymethylgroupofthe+1glycosylunit.Frand- senetal.notedthatachlorideion,apotentialmimic of superoxide or another activated oxygen species, occu- pied the fourth equatorial coordination position ofthe copperion.Substrate-bindingisassociatedwithchanges intheEPRspectrum [16,18,37], whichcouldimply that the reactivity of the copper to some extent is controlledbythepresenceofsubstrate.Ofnote,various binding studies have shown that both the presence of potentialsuperoxidemimicssuchas Cl[18] orCN [17]and reduction ofthecopper[38,39] promotesub- strate-binding.Togetherthese studiessuggest thatthe eventsleadingtoternarycomplexformationarecoupled, which is in line with conclusions derived from recent kineticstudies[30,39].

Simmons et al. reported crystal structures with xylo- oligosaccharideligandsusingthesameenzymeasFrand- sen etal. The catalyticactivityagainstxylohexaose was estimatedtobe100-foldlowercomparedtocellohexaose, andthestructuraldatashowedthattheinteractionofthe LPMOwithxylopentaosewaslessdistinct,comparedto cellopentaose (Figure 2a,b). Stacking interactions with

activesiteresidueswerenotobservedandtheconforma- tionofthecoppersiteintheenzyme–substratecomplex wasclearlydifferent(Figure2a,b),as alsoconfirmedby differencesintheEPRsignaturesofthexylohexaoseand cellohexaosecomplexes [19].

Inacombinedbiochemical,spectroscopic,andmolecular modeling study, Bissaro et al. created an experiment- supported full-scale model of an LPMO, SmAA10A, bound to a crystalline polysaccharide (chitin) surface [16] (Figure 2c,d). Importantly, themodel revealed a highly constrained active site geometry, with limited space near the copper site. The model also revealed a tunnelconnectingthebulksolventtotheactivesitethat seemed gated by a conserved second-shell glutamate, Glu60 (glutamine in some LPMOs, Figure 2). This tunnelistoonarrowforbiggermolecules,suchasascorbic acidandotherreductantstopass,whereassmallermole- culessuchasO2,O2

,H2O2orH2O,couldenterorexit.

It is worth noting that these observations add to ‘the second electron conundrum’, which entails that it is difficulttoenvisagehow thesecondelectronneeded in amonooxygenase reaction(Figure 3) would be able to reachthecatalyticcomplex[17].Whilethefirstelectron canberecruitedand storedbythenon-substratebound enzymethroughreductionofCu(II)toCu(I),itisunclear howasecondelectron, whicheitherhasto bestoredby theenzymeortimelysuppliedwhenrequired,canaccess theactive site in the LPMO–substratecomplex. Ithas beenproposedthatanelectrontransportchainorchannel wouldallow deliveryof asecondelectron [40],but this proposalisnotsupportedbyexperimentalevidencenor byconservedstructuralfeaturesacrossLPMOfamilies.

Of note, a glutamate/glutamine is pointing toward the active site in all LPMOs, and mutational studies have shownthatthisresidueisimportantforcatalysisinboth AA9andAA10LPMOs[41,42]possiblybecauseithelps in correctly positioning an oxygen species close to the active site [42]. In the recent neutron structure of NcAA9D, O’Dell et al. [22] showed evidence for an equatorially bound oxygen species interacting with His157and Gln166(whereGln166wouldbe analogous to Glu60 in SmAA10A). Similar equatorial binding was proposedbasedonaneutronstructureof JdAA10A[21].

InarecentQM/MMstudy,Caldararuetal.[43]proposed thatthesecondshellglutamateinvolvedinthislattercase (JdAA10A-Glu65) plays an important role in H2O2 for- mationbytheLPMO.Altogether,theabove-mentioned studies support the idea that this conserved Glu/Gln, which, notably, occurs at quite different positions in

(Figure1LegendContinued)clusterareindicatedbynumbers1–9.Knownadditionalsubstratespecificitiesareshownbelowthemajoractivity insmallerface.NotethatmostLPMOshaveonlybeentestedwithalimitednumberofsubstrates,sometimesonlyone.Deviatingoxidative regioselectivitiesaremarkedby**,asindicatedinthefigure.NoactivityhasyetbeenshownforAcAA10(labelledwithablueasterisk),butthe sequencewasincludedasthisisoneofthefewexamplesofaviralAA10.Theaveragemolecularweight(inkiloDalton;kDa)andnumberof aminoacids(AA)werecalculatedforeachAAfamilyusingtheprimaryAAsequencesusedtobuildthephylogenetictree.

(4)

LPMO sequences, plays an important role in LPMO catalysis by positioning [16,42,44] and/or activating [27,43]theoxygenco-substrate.

The nature ofthe co-substrateandLPMO stability

In2010,Vaaje-Kolstadetal.performedexperimentswith isotope-labeled dioxygen and water (¹⁸O2 and H218

O) leading to the conclusion that O2 is essential for the enzymereaction[3].ApartfromO2,themonooxygenase mechanism(topreactionFigure3a)requirestwoelectrons from an external electron donor. It is wellknown that H2O2 is formed in LPMO reactions because of a two- electronreductionofO2bytheelectrondonor(reductant) andbecauseoftheoxidase activityofareducedLPMO [36,45].Realizingthis,notingthatLPMOstendtobeco- expressed with H2O2 producing enzymes in fungal

secretomes[2],andpuzzledbylight-activationofLPMOs [46] and the second-electron conundrum, Bissaro et al.

assessedthepossibilitythatH2O2actsasaco-substrateof LPMOs[24].Suchaperoxygenasereactionwouldonly requireaprimingreductionoftheLPMO,afterwhichthe enzyme could perform multiple catalytic cycles when suppliedwithH2O2(Figure3b).

Indeed,Bissaroetal.[24]showedthatH2O2candrive LPMOreactionsirrespectiveofthepresence ofO2,and with consumptionof only substoichiometricamountsof reductant. Importantly, experimentsunder aerobiccon- ditions(i.e.200–250mMO2inthereactionmixture)with different concentrations of addedisotope-labeled H2O2

(H218

O2)showedthattheincorporatedoxygencamefrom H2O2evenwhentheO2concentrationwas10-foldhigher thantheH2O2concentration.Otherevidencecamefrom

Figure2

(a) (b)

(d)

4.8 Å

5.9 Å

(c)

125 Å

non-reducing end

reducing end

LPMO–substrateinteractions.

ThepicturesshowLsAA9A-cellopentaose(PDB5NLS;[19])(a),LsAA9A-xylopentaose(PDB5NLO;[19])(b),amodelofSmAA10A-

chitohexaose(c),andamodelofSmAA10Aboundtob-chitin[16](d).Thestructuresshowninpanels(a)and(b)wereobtainedfromcrystals soakedwitholigosaccharidesbeforeX-raydiffraction[19]andthemodelsofpanels(c)and(d)wereobtainedfromexperiment-guidedMD simulations[16].Thearrowsindicatetheoxidationsites;attheC4carbonofthesugarboundinthe+1subsiteforcellopentaoseand xylopentaoseboundtoLsAA9AandattheC1carbonofthesugarboundtothe1subsiteforchitohexaoseboundtoSmAA10A.Notethe increaseddistancebetweenthecopperandtheC4-carboninpanel(b)comparedtopanel(a).Axialligandsarelabeled‘ax’andareoccupied bytheC6-hydroxymethylgroup(blackstar)inpanel(a)andbyawaterinpanel(b).Theequatorialchlorideionshowninpanel(a)islabeled

‘eq’andwasobtainedfromthestructureofLsAA9Aincomplexwithcellotriose[18].Thewhitecircleandinsertinpanel(d)showthe entrancetothetunnelthatconnectstheactivesitetothebulksolvent,includingthegatekeeperresidueGlu60(correspondingtoGln162in LsAA9A)andAsn185.

(5)

the demonstration that (H2O2-consuming) horseradish peroxidaseinhibits LPMOactivity understandard conditions(1mMascorbicacidandatmosphericO2,meaning that H2O2 is generated by the system itself) [24].

Importantly, the catalytic rates obtained in H2O2 reac- tionswereordersofmagnitudehigherthatthosetypically observedinstandardO2reactions,leadingBissaroetal.to proposethatformationofH2O2isratelimitinginstandard O2-drivenreactions[4,24].Ofnote,thereisatleastone

exampleintheliteratureshowingthatanenzymeorigi- nallythoughttobeanoxidaseinfactisaperoxidase[47].

Althoughthenatureoftheco-substrateofLPMOs,O2or H2O2,remainsdebated(e.g.[48];seebelow),theclaim that LPMOs can carry out peroxygenase reactions at (unprecedented)highspeedhasbeenconfirmedinsev- eralstudies,bynumerousresearchgroupsusingmultiple LPMOs from different AA families and with different

Figure3

(a) R-H + O2 +2e-+ 2H⁺ R-OH + H2O

R-OH + H2O

no substrate

Enzyme inactivation

AscA AscA

Me-His

Substrate oxidation (C1 or C4) Substrate binding

Tyr

O2 + 2H⁺ + e-

Me monooxygenase

peroxygenase

(b)

R-H + H₂O₂

Cu(ll)

Cu(l)

His

vii

v i

iv iii

vi

ii

H2O2

H₂O₂

H₂O₂ H₂O H₂O

O₂

ReactionsinvolvedinLPMOcatalysiswithfocusonreactionsinvolvingH2O2.

Panel(a)showsanoverviewoftheO₂-driven(monooxygenase)andtheH₂O₂-driven(peroxygenase)reaction.Panel(b)showsanoverviewofkey reactionsinatypicalLPMOreaction.OxidizedLPMO,whereCu(II)interactswithanaxialandanequatorialwaterasshownhereforLsAA9A(PDB 5ACG),isreducedbyaoneelectronreduction(i)totheLPMO-Cu(I)form,wherethecopperiscoordinatedbythethreenitrogenligandsfromthe histidinebrace(PDB5ACF).H2O2willbegeneratedthroughthereactionofnon-substrate-boundLPMO-Cu(I)withO2(oxidaseactivity,ii)orfrom autoxidationoftheelectrondonor,possiblycatalyzedbytracemetals(Me)inthesolution(iii).Thereducedenzymebindstothe(poly)saccharide substrate[38]andcleavestheglycosidicbondsusingH₂O₂(orO₂)asaco-substrateinthereaction(iv).Onceprimed(i.e.reduced),andwhen usingH₂O₂,theLPMOcanperformseveralcatalyticeventswithouttheneedofbeingreducedinbetweeneachcatalyticcycles(v)[29,49].The latterislessclearfortheO₂-drivenreaction(see[63]forpossiblereactionschemes).IfareducedLPMOreactswithH₂O₂intheabsenceof substrate,orifthebindingtothesubstrateisweakorunprecise(e.g.asaresultofmutationsonthebindingsurfaceortruncationofaCBM [23,41,53]),thereactionmayleadtooxidationoftheactivesiteandinactivationoftheenzyme[24,41](vi).LPMOsproducedinfungitendtobe methylatedattheN-terminalhistidine(vii),apost-translationalmodificationthatlikelyreducesinactivationathigherH₂O₂concentrations[56].In thelowerpanels(stepv),forillustrationpurposes,thegreendotindicatestheapproximatepositionofthereactiveoxygenspeciesasderived fromthestructuresdeterminedbyFrandsenetal.[18].

(6)

substratespecificities[29,30,48,49,50–52].Thereare alsorecentstudiesshowingcorrelationsbetweentherate oftheLPMOreactionandtherateofH2O2productionin thesamereactionswithoutLPMOsubstrate[53–55].In thisrespect,Forsbergetal.havedescribedamutantofa cellulose-active AA10LPMOthatshowsreduced cellulose-degradingactivityin‘standard’reactions(i.e.withO2

and ascorbic acid),and areduced abilityto activateO2

(i.e.H2O2productionintheabsenceofsubstrate),butis asactive asthewild-typeenzymeinreactionsdrivenby exogenouslyaddedH2O2[53].

Bissaroetal.alsoshowedthattoohighconcentrationsof H2O2 leadto oxidative damage of theactive-site histi- dines,providinganexplanationforthecommonobserva- tion that LPMOs are unstable under most reaction conditions(e.g.Refs.[49,54]).Suchinactivationispre- vented by substrate binding [24]. Indeed, high substrate-concentrations [23] promote stability, whereas mutations that reduce substrate affinity of the LPMO domainitself[41,53]orthatremovebindingmodules(i.e.

CBMs)increasethesensitivityforautocatalyticinactiva- tion[23,53].Ofnote,Courtadeetal.[23]describedthe first (NMR) structure of a complete CBM-containing LPMOandcarriedoutanin-depthexperimentalassess- ment of how the CBM promotes LPMO activity and stabilityin asubstrate-dependentmanner.Experiments haveshownthatthemethylationoftheN-terminalhisti- dinefoundinfungalLPMOshaslittleeffectonenzyme functionality,butmayprovidehigherresistancetooxida- tive damage[56].

Recentinsights fromkinetic studiesusing O2

or H₂O₂as co-substrate

ReportedcatalyticratesforLPMOsundertypicalreaction conditions(atmosphericO2,1mMreductant)tendtobein theorderof0.1s¹or(much)lower,asrecentlyreviewedby Bissaroetal.[4].KineticcharacterizationofanAA9LPMO actingoncellotetraosewithO2astheco-substrateyieldeda kcatof0.11s¹,aKmof43mMwithrespecttothecarbohy- dratesubstrateandakcat/Kmof 2.610³M¹s¹[18].

Thefirstcomprehensivekineticcharacterizationofa(chitin-active)LPMOwithH2O2astheco-substrateyielded quite differentvalues: akcatof 6.7s¹and Kmvaluesof 0.58gL¹and 2.8mM for chitinand H2O2,respectively [30].Theresultingkcat/Kmis210⁶M¹s¹forH2O2, whichisinthesameorderofmagnitudeasreportedkcat/Km

valuesforperoxygenases[57,58].

Hangasky et al. studied oxidation of cellohexaose by a fungalAA9LPMOwithO2orH2O2asco-substrateandin thepresenceof2mMascorbicacid[48].Reactionswith O2,ataconcentrationof208mM,andvaryingconcentra- tionsofcellohexaose,yieldedanapparentkcatof0.17s¹ and a Km of 32mM with respect to cellohexaose. This yieldsakcat/Kmof510³M¹s¹,whichissimilartothe value obtained by Frandsen et al. ([18]; discussed

above). Reactions with aconstantcellohexaose concentration(1mM)andvaryingO2concentrations(0–800mM) yielded anapparentkcatof0.28s¹andaKmof230mM with respect to O2, which corresponds to a kcat/Km of 110³M¹s¹.Ofnote,thisvalueisaboutthreeorders of magnitude lower than the kcat/Km determined by Kuusk et al. [30] for H2O2-driven chitin conversion.

Accordingly, reactions with H2O2 in the range from 12.5 to 100mM by Hangasky et al. [48] yielded rate constants between 4 to 15s¹. Although, the authors didnot calculateaKm withrespectto H2O2,thisvalue canbeestimatedthroughaMichaelis–Mentenanalysisof data in Table S9[48], yielding aK^m of 53mM,which leads toanestimated kcat/Kmof 310⁵M¹s¹. A crucial differencebetweenthe O2and H2O2mecha- nismconcernstheneedforareductant.WhileintheO2

mechanism,thereductantisconsumedstoichiometrically with product formation, the H2O2 mechanism only requires a ‘priming’ reduction, and more reductant is onlyneeded uponoccasionalre-oxidationoftheLPMO (Figure 3). Still, the H2O2 mechanism does require reducingpower,whichindeedmaybecomerate-limiting under certain conditions [24]. It is well known that LPMO activity is reductant-dependent [2,59] but it is less clear why and how. Unravelling the role of the reductantisnotstraightforwardbecause,nexttoLPMO reduction, the reductant will also affect the enzyme- dependent and enzyme-independent generation of H2O2.Thesecomplicationshaverecentlybeenunraveled inkineticstudiesofachitin-activeLPMObyKuusketal.

[29]whoshowedthat,oncereduced,theLPMOcarried out18oxidativecleavagesusingH2O2asco-substrate.

FurtherillustratingtheimportanceofH2O2,Bissaroetal.

and Mu¨ller et al. showed that the LPMO activity in a commercial cellulase cocktailacting onAvicel couldbe increasedbyuptotwoordersofmagnitudeinanaerobic reactionswithH2O2feeding,comparedtostandardaero- bicreactionsutilizingO2andstoichiometricamountsof reductant [24,49]. Calculations showed that, under optimalconditions, eachLPMOin thereactionmixture catalyzedatleast1500peroxygenationreactionswhilethe ratio between reactions catalyzed and reductant con- sumed wasintheorderof15:1.Thisapplied studyalso underpinned the risk of enzyme inactivation by

‘overfeeding’H2O2(Figure3b).

Allinall,accumulatingkineticdataindicatesthatH2O2is the preferredco-substrate of LPMOsand that theper- oxygenasereactioncanreachmuchhigherratesthanthe very low rates observed for O2-driven reactions. Of course, as pointed out by Hangasky et al. [48], and assuming that the monooxygenase reaction does occur at all (see below for discussion), what will happen in naturedependsontheconcentrationsof O2,H2O2,and reductant.

(7)

Recentinsightsfrommodeling usingO₂or H2O2as co-substrate

ThediscoverythatLPMOscontainasingle coppersite withoutanyapparentadditionalredoxcofactortriggered thecuriosityofthemetalloenzymecommunity.Though several LPMOs have been subjected to spectroscopic methods, no reaction intermediates have yet been discovered.Thus,mechanisticinsightatatomistic/molecular levelhasmainlybeengainedfromcomputationalefforts thathaveexploredpossiblereactionpathways.Inacom- binedspectroscopicandcomputationalstudy,Kjaergaard etal.[60]demonstratedformationofacoppersuperoxide complex, [CuOO]⁺, when a reduced LPMO interacts

withO2in theabsenceofsubstrate.Itseems,however, questionable whether superoxide is strong enough to abstractahydrogenfromacarboninthescissileglycosidic bond, most studies conclude that a stronger oxidative speciesisneeded [25,26,27,28,61,62].

The first computational study to address the catalytic mechanism of LPMOs favored a copper oxyl, [CuO]⁺, intermediateasthereactivespecies[61].Thishydrogen atomabstraction[CuO]⁺speciesappearsinmostcompu- tationalstudiesandcouldalsobeacopper-oxoorproton- ated[CuOH]²⁺species.Importantly,whileKimetal.[61]

considered axial binding of O2, there is now abundant

Figure4

(ll-a)

(lll-c)

(lll-b)

(l) (ll)

(lll)

(lll -a)

homolytic cleavage

ll-a

PutativereactionmechanismsforpolysaccharideoxidationbyLPMOsusingH2O2(leftside)orO2(rightside)asco-substrate.

Reducedcopper,andreducingequivalentsarecoloredblue,oxidizedcopperandoxygenspeciesarecoloredorangeandred,respectively.H2O2

reactswiththeCu(I)centerleadingtotheproductionofahydroxylradicalviahomolyticbondcleavage(pathwayI)orviaabase-assisted mechanism(pathwayII).Theprotonthatisheldbytheputativebasecanreacteitherwiththecopper-boundoxygenatom(pathwayII-a,grey)or withtheleavinghydroxidegroup(pathwayII-b,magenta),whichleadstoeliminationofawatermoleculeandformationofacopper-oxyl intermediate.Pathways(I)and(II-a)bothleadtotheformationofaCu(II)-hydroxideintermediateandahydroxylradical.Thishydroxylradical catalyzeshydrogenatomabstraction(haa)eitherfromtheCu(II)-hydroxide(haa1)orfromthesubstrate(haa1’).TheformerscenarioleadstoaCu (II)-oxylintermediatethatcancatalyzeHAAonthesubstrate(haa2).Inbothcases(haa1+haa2orhaa1’),awatermoleculeiseliminatedanda substrateradical(R)andacommonCu(II)–OHintermediatearegenerated.TheCu(II)-associatedhydroxidemergeswiththesubstrateradical throughareboundmechanism,leadingtohydroxylationofthesubstrateandregenerationoftheCu(I)center,whichcanenteranewcatalytic cycle.ThemechanismanalyzedindetailbyWangetal.ishighlightedbygreydots[27].IntheO2-basedmechanisms(rightsideofthefigure),the pathwayshavebeenexaminedinthefollowingcomputationalstudies;(III-a)[25,26,60],III-b[28,61].Thisfigureisadaptedfromthe

supplementaryinformationofBissaroetal.[24].

(8)

computational and structural evidence that the oxygen co-substrate binds the equatorial position (Figure 2a) [18,21,22,44]. Bertini et al. [26] obtained a [CuO]⁺ complex displaying a distorted tetrahedral symmetry, resulting in an in-between axial and equatorial oxygen atomposition. Thefirstcomputationalreportonareac- tionmechanisminvolvingH2O2asaLPMOco-substrate supportedone ofthepotentialmechanismsput forward byBissaroetal.[24]thatimpliesformationofahydroxyl radicalandacopper-associatedhydroxideupontheH2O2

reaction with LPMO-Cu(I) (see Figure 4) [27]. The calculationsbyWangetal.showedthatthehighlyreactive hydroxylradicalsubsequentlyabstractsahydrogenatom from the copper-associated hydroxide, resulting in a [CuO]⁺ species. Finally, the[CuO]⁺ speciesabstracts a hydrogenatomfromtheoligosaccharidesubstratebefore it recombines with the substrate radical, in a rebound mechanism[61],yieldingahydroxylatedproduct.Essen- tiallysimilarconclusionswereobtainedbyHedega˚rdand Ryde [28]; however, the two reports do not agree on intermediate spin states, which is an interesting detail thatdeservesfurtherattention.

Although the [CuO]⁺ state regularly appears in the LPMO literature, it must be emphasized that several alternatives have beenproposed for theoxygenspecies that abstracts the hydrogen from the substrate. The nature ofthe trueintermediate reactive-oxygenspecies has not been experimentally determined and, despite recentmodelingstudies,multipleplausiblemechanisms remain. Figure 4 shows prevalentpossible mechanisms for both O2 and H2O2-driven reactions. We refer to WaltonandDavies[63]andMeieretal.[62]forcompre- hensivereviews ofmechanisticaspectsof LPMOs.

Propercomplexformationbetweenenzymeandsubstrate iscrucialfor LPMOfunctionalityandisof greatimpor- tance in both experimental and computational assess- ment of LPMO reactivity. Simmons et al. [19] have shown thatvariation in substratepositioningmay occur (Figure2a,b)andthisobservation,combinedwith(rather limited)studiesofcatalyticactivityledtheseauthorsto suggest that the catalytic mechanism employed by LPMOs may vary, depending on the substrate. We believe thatit istoo earlyto conclude thatone LPMO mayhavemultiplelegitimatecatalyticmechanisms(see alsobelow),butthiscannotbeexcluded.Moreingeneral, there couldbemechanisticvariationsamongLPMOs.

Conclusions

Despite years of intensive research, several LPMO secretsremaintoberesolved.Researchontheseenzymes iscomplicatedbytheinsolubilityofwhatlikelyaretheir naturalandindustriallymostrelevantsubstrates.Experi- mentalverificationofthereactiveoxygenintermediateis complicated by the fact that binding of the substrate helpsinshapingthegeometryandreactivityofthecopper

site,meaningthatstudiesonenzymesintheabsenceof (appropriate) substrate can only tell part of the story.

Another complication liesin the multitude of reactions that mayoccur inLPMO reactions, includingautocata- lytic inactivation of the LPMO, potential depletion of reductant,andbothproductionandconsumptionofH2O2

throughavarietyofprocesses(Figure3b;seealsoBissaro etal.[4]forfurtherdiscussions).Indeed,kineticdatafor LPMOsisscarce,andproducingsuchdataisexperimen- tallychallenging.

Lack of kinetic data may be the underlying reason to someofthecurrentuncertaintiesinthefield.Wewould arguethatseeminglycontradictoryresultsmayinpartbe due to the fact that some reports make quantitative statementsaboutLPMOactivitythatarebasedonsingle time-pointmeasurements.Becauseoftheinactivationof LPMOsovertime,toanextentthisisdependentonthe reaction conditions, including the (varying) substrate concentration, quantitative statements based on single time point measurements are riskyat best and in most cases notvalid.The readerisreferredtoForsberg etal.

[53] for an example illustrating this point. While the suggestion by Simmons et al. [19] that LPMOs may usemultiplereactionmechanismsmayverywellbetrue, this suggestion is based on single time point measurements of LPMO activity. Likewise, the suggestion by Hangaskyetal.[48]thatLPMOsdouseO2directly(next toH2O2)mayverywellbetrue,but,inouropinion,more detailed kinetic analysis is needed to substantiate this conclusion (seebelow).

TheimpactofsubstrateconcentrationonLPMOstability isofcrucialimportance[23],notintheleastinapplied settings where LPMOsmay become inactivated as the substratebecomesdepleted[49],thatisatatimepoint duringtheprocesswheretheymaybeparticularlyneeded fordegradingtheremaining,potentiallymostrecalcitrant, material. Ofnote,thedetailedkineticstudiesbyKuusk etal.[30]showedthattherateofauto-catalyticLPMO inactivationintheabsenceofsubstrateisthreeordersof magnitude lower that the rate of substrate cleavage (under substrate saturating conditions). So, as long as substrate concentrations are high, autocatalytic LPMO inactivationislargelyprevented.

While therolesofO2andH2O2asco-substrates remain somewhat controversial, it is now widely accepted that H2O2isabonafideco-substratethatyieldshighLPMO catalyticrates.IthasbeenclaimedthatLPMOsbecome lessspecific andlessstable whenfueledbyH2O2[48], but this claim is not substantiated by other studies, includingourownpublishedandunpublisheddata.Sta- bility and specificity issues may occur, but these are affected by the reaction conditions (e.g. the LPMO–

substrate-reductant-O2/H2O2ratio)andnotbythenature of theco-substrate, O2orH2O2,as such.

(9)

Although it cannot be excluded that O2 can be used directly as co-substrate, possibly via formation of a H2O2molecule thatneverleaves theenzyme substrate complex [43,55], we would argue that current kinetic evidenceisthin.Forexample,researchershaveobserved that under some conditions, neithercatalase norhorse- radishperoxidaseinhibitLPMOs[48,64],whichmaybe taken to imply that free H2O2 does not play a role in catalysis. However, an equally plausible explanation would be that, under the conditions used, the LPMO effectivelycompeteswith theseother H2O2-consuming enzymes,as discussedfor catalasebyKuusketal.[29].

Thereisagreatneedfordevelopmentofreliable,easy-to useassaysofLPMOactivity,whichtakeintoaccountall ormostcomplexitiesdiscussedaboveandwhich,prefer- ably, should address theformation of both soluble and insoluble oxidized products. Despiteprogress in recent years[51,52,65],more workiscertainlyneeded.

Thepastyearshaveshownmassiveprogressanditseems likelythatseveralopenquestionswillbeansweredinthe near future, while new LPMO functionalities may be discovered.Oneof themostimportant developmentsis theinsightintotheroleofsecondshellresidues,suchas the Glu/Gln (discussed above), in positioning and/or activatingtheoxygenco-substrate[16,21,22,28,42,43].

Perhaps existing LPMO mutants, or novel mutants designed basedon thecurrentinsights,may eventually allowdetectionandcharacterizationofrelevantreaction intermediates. Generally, more extensive mutagenesis studies,including,mostimportantly,properkineticchar- acterizationofeachenzymevariant,areessentialforthe field.Moreadvancedanddetailedstudiesoftheinterac- tionbetweenLPMOsandtheirpolymericsubstrates(e.g.

Ref.[10]),which,notably,includesolublepolymerssuch asxyloglucan,arealso greatlyrequired.

Conflictofintereststatement Nothingdeclared.

Acknowledgements

Wethankcurrentandformerteammembersandcollaboratorsfortheir contributionstoourLPMOresearch,whichhasbeensupportedbythe ResearchCouncilofNorway,mostrecentlythroughgrants240967,243663, 243950,257622,256766,268002,262853,221576and226244.Additional supportwasreceivedfromtheMarie-CurieFP7COFUNDPeople Programme,throughtheawardofanAgreenSkillsfellowship(undergrant agreementn267196),andfromtheFrenchInstitutNationaldela RechercheAgronomique(INRA).

References andrecommendedreading

Papersofparticularinterest,publishedwithintheperiodofreview, havebeenhighlightedas:

ofspecialinterest ofoutstandinginterest

1. ErikssonKE,PetterssonB,WestermarkU:Oxidation:an importantenzymereactioninfungaldegradationofcellulose.

FEBSLett1974,49:282-285.

2. KracherD,ScheiblbrandnerS,FeliceAK,BreslmayrE,PreimsM, LudwickaK,HaltrichD,EijsinkVGH,LudwigR:Extracellular electrontransfersystemsfuelcelluloseoxidativedegradation.

Science2016,352:1098-1101.

3. Vaaje-KolstadG,WesterengB,HornSJ,LiuZ,ZhaiH,SørlieM, EijsinkVGH:Anoxidativeenzymeboostingtheenzymatic conversionofrecalcitrantpolysaccharides.Science2010, 330:219-222.

4.

BissaroB,Va´rnaiA,RøhrA˚K,EijsinkVGH:Oxidoreductasesand reactiveoxygenspeciesinconversionoflignocellulosic biomass.MicrobiolMolBiolRev2018,82http://dx.doi.org/

10.1128/MMBR.00029-00018.

Comprehensivereviewofoxidoreductasesthatareputativelyinvolvedin fungalbiomassconversion,includingacriticalassessmentofexisting LPMOdatainlightofthenewlydiscoveredperoxygenaseactivity,and withanoverviewofavailablekineticdata.

5. Su¨tzlL,LaurentC,AbreraAT,Schu¨tzG,LudwigR,HaltrichD:

MultiplicityofenzymaticfunctionsintheCAZyAA3family.

ApplMicrobiolBiotechnol2018,102:2477-2492.

6. WestermarkU,ErikssonKE:Cellobiose-quinone

oxidoreductase,anewwood-degradingenzymefromwhite- rotfungi.ActaChemScandB1974,B28:209-214.

7. HenrikssonG,JohanssonG,PetterssonG:Acriticalreviewof cellobiosedehydrogenases.JBiotechnol2000,78:93-113.

8. PhillipsCM,BeesonWT,CateJH,MarlettaMA:Cellobiose dehydrogenaseandacopper-dependentpolysaccharide monooxygenasepotentiatecellulosedegradationby Neurosporacrassa.ACSChemBiol2011,6:1399-1406.

9. QuinlanRJ,SweeneyMD,LoLeggioL,OttenH,PoulsenJC, JohansenKS,KroghKB,JørgensenCI,TovborgM,AnthonsenA etal.:Insightsintotheoxidativedegradationofcellulosebya coppermetalloenzymethatexploitsbiomasscomponents.

ProcNatlAcadSciUSA2011,108:15079-15084.

10. EibingerM,SattelkowJ,GannerT,PlankH,NidetzkyB:Single- moleculestudyofoxidativeenzymaticdeconstructionof cellulose.NatCommun2017,8:894.

11. VermaasJV,CrowleyMF,BeckhamGT,PayneCM:Effectsof lyticpolysaccharidemonooxygenaseoxidationoncellulose structureandbindingofoxidizedcelluloseoligomersto cellulases.JPhysChemB2015,119:6129-6143.

12. VillaresA,MoreauC,Bennati-GranierC,GarajovaS,FoucatL, FalourdX,SaakeB,BerrinJG,CathalaB:Lyticpolysaccharide monooxygenasesdisruptthecellulosefibersstructure.Sci Rep2017,7:40262.

13. LenfantN,HainautM,TerraponN,DrulaE,LombardV, HenrissatB:Abioinformaticsanalysisof3400lytic

polysaccharideoxidasesfromfamilyAA9.CarbohydrRes2017, 448:166-174.

14. SabbadinF,HemsworthGR,CianoL,HenrissatB,DupreeP, TryfonaT,MarquesRDS,SweeneyST,BesserK,EliasLetal.:An ancientfamilyoflyticpolysaccharidemonooxygenaseswith rolesinarthropoddevelopmentandbiomassdigestion.Nat Commun2018,9:756.

ThispaperdescribesthediscoveryofanewLPMOfamily(AA15)which expandsthepresenceofLPMOsfrommainlymicrobialtotheanimal kingdom.

15. CouturierM,LadevezeS,SulzenbacherG,CianoL,FanuelM, MoreauC,VillaresA,CathalaB,ChaspoulF,FrandsenKEetal.:

Lyticxylanoxidasesfromwood-decayfungiunlockbiomass degradation.NatChemBiol2018,14:306-310.

ThisstudydescribesanewLPMOfamily(AA14)actingspecificallyonco- polymericassembliesofcelluloseandxylan;thisnovelLPMOwasshown topromote theefficiencyofacellulolyticenzymecocktailinbiomass conversion,likelybypromotingthedissociationofhemicellulosefrom cellulose.

16.

BissaroB,IsaksenI,Vaaje-KolstadG,EijsinkVGH,RøhrA˚K:How alyticpolysaccharidemonooxygenasebindscrystalline chitin.Biochemistry2018,57:1893-1906.

Experiment-guidedMDsimulationsshowinghow anAA10bindstoa crystallinesubstrateandthepotentialinvolvementofaconservedsecond shellGlu/Glnintransportingdioxygenspecies totheactivesitewhen boundtothesubstrate.