Beyond active site residues: overall structural dynamics control catalysis in flavin-containing and heme-containing monooxygenases

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University of Groningen

Beyond active site residues

Fürst, Maximilian J. L. J.; Fiorentini, Filippo; Fraaije, Marco W.

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Current Opinion in Structural Biology DOI:

10.1016/j.sbi.2019.01.019

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Fürst, M. J. L. J., Fiorentini, F., & Fraaije, M. W. (2019). Beyond active site residues: overall structural dynamics control catalysis in flavin-containing and heme-containing monooxygenases. Current Opinion in Structural Biology, 59, 29-37. https://doi.org/10.1016/j.sbi.2019.01.019

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Beyond

active

site

residues:

overall

structural

dynamics

control

catalysis

in

flavin-containing

and

heme-containing

monooxygenases

Maximilian

JLJ

Fu¨rst

1

,

Filippo

Fiorentini

2

and

Marco

W

Fraaije

1 Monooxygenases(MOs)facethechallengingreactionofan

organictarget,oxygenandacofactor–mostcommonlyheme orflavin.Tocorrectlychoreographthesubstratesspatiallyand temporally,MOsevolvedavarietyofstrategies,whichinvolve structuralflexibility.Besidesclassicaldomainandloop movements,flavin-containingMOsfeatureconformational changesoftheirflavinprostheticgroupandtheirnicotinamide cofactor.Withsimilarmechanismsemerginginvarious subclasses,theirgeneralityandinvolvementinselectivityare intriguingquestions.CytochromeP450MOsareoften inherentlyplasticandlargemovementsofindividualsegments throughouttheentirestructureoccur.Asthesecomplicated andoftenunpredictablemovementsarelargelyresponsiblefor substrateuptake,engineeringstrategiesfortheseenzymes weremostlysuccessfulwhenrandomlymutatingresidues acrosstheentirestructure.

Addresses

1_Molecular_Enzymology_Group,_University_of_Groningen,_Nijenborgh_4,

9747AG,Groningen,TheNetherlands

2_Department_of_Biology_and_{Biotechnology,}_University_of_Pavia,_Via

Ferrata1,27100,Pavia,Italy

Correspondingauthor:Fraaije,MarcoW(m.w.fraaije@rug.nl)

CurrentOpinioninStructuralBiology2019,59:29–37

ThisreviewcomesfromathemedissueonCatalysisandregulation EditedbyPhilipColeandAndreaMattevi

https://doi.org/10.1016/j.sbi.2019.01.019

0959-440X/ã2018ElsevierLtd.Allrightsreserved.

Introduction

AerobiclifeevolvedtouseO2asanelectronacceptorinthe

respiratorychainandasaco-substratetooxygenateorganic compounds using enzymes such as monooxygenases (MOs). As the spin-forbidden reaction of tripletground state O2 with singlet organic compounds is very slow,

enzymeslowertheenergybarrierbyreductivelyactivating oxygen.Unlesstheorganicsubstrateprovidesthereducing power,thisreactionrequiresacofactor.Open-shell transi-tionmetalssuchascopperorironcanbedeployed,andthe latter isoftencomplexedbyaporphyrinscaffold—the

heme cofactor. Alternatively, MOsuse a purely organic flavinmononucleotide(FMN)orflavinadenine dinucleo-tide(FAD)cofactor.IntheseveralhundredavailableMO structures,thetwomostfrequentlyco-crystallizedligands are heme (43%) and FAD (14%), which are used by cytochromeP450MOs(CYPsorP450s)andflavoprotein MOs,respectively.Thetraditionalcenterofattentionwas theactivesiteoftheMOs,whichprovidesthestructural context for facilitating catalysis — electron transfer, O2

activation,andoxygenation.However,ifanystatic struc-ture is insufficient in describing an enzyme’s mode of action, this is especially true with MOs due to their extremely dynamicnature (Figure 1).For afull under-standingofthereactionofMOs,weneedtolookbeyond thesupposedcatalyticcenter.

Flavoprotein

monooxygenases

The isoalloxazine ring enables flavins to stabilize and shuttlebetweenredoxstates.InflavoproteinMOs,oxygen is activated by the transfer of one electron from fully reducedflavintoO2,followedbythecouplingofthecaged

radicalpairattheflavin’sC4alocus[1].Characteristically, flavoproteinMOsstabilizetheresultingcatalytic(hydro) peroxyflavin [2].Theelectronsoriginatefromareduced nicotinamidecofactor–NAD(P)H–whichcanbindeither transientlyorpermanently.Theformeristhecaseforthe aromatichydroxylasesofclassAflavoproteinMOs,where the nicotinamide cofactor dissociates immediately after reducingamobileflavin[3,4](Figure1).Theseenzymes arequitenarrowinsubstratescopeand‘cautious’:before NAD(P)Hisconsumed,apotentialsubstrateneedstobe ‘proofread’ [5].Incontrast, NAD(P)Hisconsumed sub-strate-independentlyandboundinvariousconformations throughoutthecatalyticcyclein‘bold’classBflavoprotein MOs(Figure 1). These compriseN-hydroxylatingMOs (NMOs),whicharehighlysubstrate-specific, heteroatom-oxygenatingflavin-containingMOs(FMOs),andketoneto ester-transformingBaeyer-VilligerMOs(BVMOs),which oftenshowrelaxedsubstratescopes.

Mobileflavins

For the prototype class A flavoprotein MO, p-hydroxy-benzoate hydroxylase, a delicate dynamic interplay between the coenzyme NADPH and the prosthetic FAD cofactor, has been unraveled [6]. For reduction, the flavin of class A MOs swings toward NADPH into an‘out’positionusingtheribitylcarbonsaspivotpoints (Figure2a).Next,NADP+isreleased,FADreturnstothe

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‘in’position[7],and theformed C4a-hydroperoxyflavin hydroxylatesthesubstratethroughelectrophilicaromatic substitution.Whilethis mechanismwaselucidated dec-ades ago [3,4], its clinical relevance was established recently, whenabacterial tetracycline MOthatconfers antibioticresistancewasshowntobeefficientlyinhibited by asubstrate analogue, which locks FAD in the ‘out’

position [8]. Furthermore, novel variations on the mobile flavin mechanism were discovered in two para-logous class A MOs converting the same multicyclic substratetodivergentproductsinabifurcatingmetabolic pathway [9]. While one, RebC, substitutes a carboxyl group with a carbonyl group, the second, StaC, only decarboxylates. Apparently, RebC uses flavin mobility

30 Catalysisandregulation

Figure1

Cytochrome P450 MOs

resng state substrate binding reducon oxygen acvaon substrate oxidaon elecon transfer oxygen transfer

Class A MOs

Class B MOs

Current Opinion in Structural Biology

Simplifiedand/orexemplarymechanismofMOclassesandstructuralflexibility.P450sareinherentlyplastic,withflexibleregionsoccurring throughouttheproteinstructure.ClassAflavoproteinMOsarewell-knownfortheirmobileflavincofactor,whereasinclassB,oftentheNAD(P) cofactorisfoundinvariousconformations.

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for reduction before hydroxylating the substrate’s enol tautomer, while StaC’s mobile flavin accelerates the spontaneousdecarboxylationof theketo tautomervia a steric and/or electrostatic clash. The same group also discovered thatmobileflavinsoccurin N-hydroxylating MOsof classB[10].

AnearlyindicationforaconformationalchangeinNMOs wastheproposedallostericregulation[11]ofL-ornithine

MO(SidA)byL-arginine[12].However,theregulationis

likelyrather acompetitiveinhibition, asstructureslater revealedL-argininetobindatthesame positioninSidA

[13] as L-ornithinein a homologous NMO (PvdA)[14].

Eventually,structuresofanotherhomolog(KtzI)showed FADtoundergoconformationalchanges[10].Whilethe swing of the flavinin class AMOsoccursnearly in the planeoftheisoalloxazinering,KtzI’sflavinpivotslargely attheribitylC1androtatesoutoftheplane(Figure2b). Asthistrajectoryclasheswiththenicotinamideriboside, itmightrepresentanNADP+ejectionmechanism.Inthe resting state,theoxidizedflavinis probablyinan equi-libriumbetween ‘in’and ‘out’.Nohydridetransfer ori-entationwasobserved,butreducedflavinwasalways‘in’ and thehydroperoxyflavinlikelyretains thisposition. A distorted nicotinamide incrystals of PvdAtrapped with theproductsuggestedaninitialdestabilizationofNADP+ [14],whichthenwouldbeejectedbythemovingflavin.

Mobilenicotinamidecofactors

As they bind NADP stably [2], class B MOs are often crystallizedincomplexwithbothcofactors.Several orien-tationsofNADPcanbeobservedinavailablestructures. With varying degrees of confidence, these have been attributedtothedualroleofthecofactoroverthecourse of the catalytic cycle: reduction of the flavin and

stabilization of the (hydro)peroxyflavin [2]. As the two roles require different orientations and no structure appropriate for hydride transfer is known, a ‘sliding mechanism’hasbeenproposed[15](Figure3a). Accord-ingly,NADPH reducestheflavinwhilesliding overthe isoalloxazine into its fixed and commonly observed ‘stabilization’position.Variousstructuresappeartoshow the positions sampled on the way: stacked above the flavin in steroidMO (STMO,PDBIDs4AOS), and an intermediate positionin one crystal form of cyclohexa-none MO (CHMO, PDB ID 3GWF). Problematically, however,themodelconflictswithexperimentsshowing thatNADPH’spro-Rhydridereducestheflavin,whichis incompatible with the anti conformation of the flavin-stacked NADPH observed in the before-mentioned structures.Althoughthestereoselectivitycanbealtered byactivesitemutagenesis,itisconservedthroughoutthe classBMOs[16].TwoexceptionsinthePDBdisplaya moresuitablesynconformation:cadaverineMO(PDBID 5O8R[17])whereunfortunatelytheNADPwasmodeled ondiffuseelectrondensityanditsvalidityisdoubtful;and amutantofabacterialtrimethyl-amineMO(TMM,PDB ID5IQ4[18]),wheretheelectrondensityofthe nicotin-amidesuffered fromlowoccupancy(Figure3b–c).

WhenNADP+isinits‘usual’position,ahydrogenbond fromtheamideoxygencruciallystabilizesthe N5hydrogen of thereducedflavin[18]andthe subsequentlyforming peroxyflavin [19]. Additionally, the ribose20hydroxylgroup hydrogenbondstothereactionintermediateinBVMOs, and donatesitsproton to formthehydroperoxyflavin in FMOs/NMOs[20].Byaflipoftheamide,theaminecan alsointeractwiththeN5oftheoxidizedflavinafterproduct formation in a retained overall conformation of NADP+.Thedistinctionisdifficult,astheorientationof

(a) (b)

Mobileflavincofactors.(a)TheflavinoftheclassAflavoproteinMOp-hydroxy-benzoatehydroxylaseswingsintheplaneoftheisoalloxazinering froman‘in’position(greycarbons,1PBE),toan‘out’position(1DOD,yellowcarbons).Thesubstrate(violetcarbons)andacut-opensurfaceof theproteinstemsfrom1PBE.(b)OverlayoftheclassBL-ornithineMO(KtzI)incomplexwithL-ornithine,‘in’FAD,andNADP+_(violet,_white,_and

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theamidecanusually notbeinferredfromtheelectron density.Theflexible partofNADP isthenicotinamide mononucleotide.Ahydrogenbondbetweenitsphosphate andaconserved,hydroxyl-containingaminoacid[21]isthe pivot point linking it to the well-anchored adenosine mononucleotidemoiety. Thiswasalsoobservedfor two additionalNADP+orientations,whichfeaturearotatedanti nicotinamide riboside.Ahalfrotationoccurredincrystalloin TMMuponsubstratesoaking(PDBID5GSN[18]),andin abacterialmFMOupondisruptionofeitheroftwo hydro-genbondstothenicotinamide:fromtheNADP+amineto theflavinN5(usinganNADPanalog,PDBID2XLT)or from the ribose to a central asparagine (in an aspartate mutant,PDBID 2XLR) [22](Figure 3a). Interestingly, aspartate is the conserved residue in BVMOs, which, although never observed with the half-rotated cofactor, deliveredthe onlystructurewithafully-rotatedNADP+ [23](PDBID3UCL,Figure3a).Inthisstructure,asinthe half-rotatedTMMstructure,additionalelectrondensityon top of the flavin was assigned to substrate molecules.

However,thisassignmentiscontroversial,asitstands in contrasttopreviousligandpositionsandthereisa notice-ableconnectiontothedensityofthenicotinamideriboside (Figure3d–e).Itcan,therefore,hardlybeexcludedthatthe originisanalternativeconformation ofNADP,ratherthana ligand.Furtherresearchshouldclarifythesubstrate posi-tionandwhethertherotatedcofactorisageneral mecha-nismoftheenzymeclass.Thismaycontributetosolving tworemainingpuzzles:thestructuralbasisforthedifferent mechanisms and reactivities, and the cause of the vast discrepancyinsubstratespecificity.

MobilityofloopsanddomainsinflavoproteinMOs

Substrateacceptanceisanintenselyresearchedenzyme trait with biotechnological relevance, and protein flexi-bilitywasidentifiedas‘perhapsthesinglemostimportant mechanism’toachievepromiscuity[24].Themost flexi-bleprotein structuresare loops and unsurprisingly, this structuralelementdiffersmostamongotherwisesimilar flavoproteinMOs.

Figure3 (a) (b) (c) (e) (d)

L

N2

N1

NADPandproteinmobility.(a)Cut-opensurfaceofPAMO(1W4X)withFAD-(yellow)-,NADP-(blue)andhelicaldomains(orange).An‘L’marksa movingBVMOloopwithaconservedtryptophan(lightgrey),whichcanbefoldedin(2YLR,whitecartoon)whenNADP+_is_present_or_form_a

b-hairpin(3UOZ,darkgrey)inahomolog.Theinsetmagnifiestheflavin(yellowcarbons)andthevariouspositionsfoundinclassBMOsof NADP’snicotinamidering.‘N1’markstheapparent‘sliding’movementbyoverlayingSTMO(4AOS,greencarbons),CHMO(3GWF,cyancarbons), andPAMO(2YLR,bluecarbons).‘N2’marksanapparentrotationviaahalf-rotated(TMM,5GSN,darkvioletcarbonsandmFMO,2XLR,violet carbons)toafullyrotatedforminCHMO(3UCL,pinkcarbons).(b–e)Electrondensities(s=1)ofstructureswithcontroversialNADP+_modeling:_(b)

cadaverineMO(5O8R)and(c)theTMMY207Smutant(5IQ4)aremodeledwithNADPinahydridetransfer-suitablesynconformation,butsuffer frompoorelectrondensityatthenicotinamideend.(d)CHMO(3UCL)and(e)TMM(5GSN)withhalf-rotated,andfullyrotatedNADP+_,_{respectively,}

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closeandactasahinge[25])appearscrucialforfunction andwascalled‘controlloop’[26].Ifvisible,theloopfolds ontopoftheRossmannfold-boundNADP,therebyoften trappingthecofactorinthecrystalstructure(Figure3a). SAXSexperimentsindicatethatNADP+exposurefavors this folded state, which also coincides with ‘closed’ enzymeconformations.In‘open’conformations,notonly thedisorderedloopmaybeunstructured,butalsoawide swingintothesolvent(deemedacrystallizationartefact) wasseeninphenylacetoneMO(PAMO,PDBID1W4X [27]), and 2-oxo-D3 -4,5,5-trimethylcyclopentenylacetyl-coenzyme A MO, where the loop adopts a structured b-hairpin(e.g.PDBID3UOZ[28])(Figure3a).Acentral role in loop reorganization is assumed for a conserved tryptophan(Figure3a),whichisanactivesiteresidueif theloopisfoldedandwhoseremovaldrasticallyreduces enzymeactivity[15].Theloopmayalsoactasan‘atomic switch’ [15,26] that connects the active site and the BVMOsignature motif[29],astrictlyconserved stretch attheedgeof theNADPdomain,inexplicablyfarfrom the active site. A histidine in this motif adopts varying conformations and can form contacts with the linker region, which in turn is connected to the control loop [15]. Theimportanceand ability ofthe linkerfor long-range effects became also apparent when mutations in this region drastically altered enzymatic activity [30]. ConsideringthattheSAXSresultswerenotfully explain-able by loop movements, these data collectively sug-gestedthatlargermovementsofthedomainscouldoccur. Domain rotations of up to 6 [15,31] were already observed, but the extent might have been artificially hindered bycrystalpacking[26].Adrasticdomain rota-tionof30hasbeenobservedforanNMO,NbtG[32],but itisunknownwhetherotherNMOs,letaloneotherclass B families can sample this conformation as well. More distantly related enzymes with thesame domain archi-tecture are able to rotate by even 67 [33], and some members of class A flavoprotein MOs can cover their active sitewith aflexible ‘lid’ domain [34].Future dis-coveries on such mechanisms in class B MOs can be expected, andthesemaybekey inunderstanding their varying selectivities. It might also allow to explain the profoundallostericeffectsofactivesite-remotemutations [35], and the surprisingly mild effects of removal of residues that(seemingly)formtheactive site[36].

Cytochrome

P450s

Referred to as ‘nature’s blowtorch’ [37], the iron-oxo species forming in the core of cytochrome P450s MOs (P450s)areendowedwiththeoxidativepowertocatalyze variousreactions:besidesperformingdealkylations, het-eroatom oxidationsandepoxidations,P450shydroxylate non-activated C–H and C–C bonds in substrates of diverse size,functional group composition, andpolarity [38]. Similar to class A flavoprotein MOs, the catalytic mechanismisinitiatedbysubstratebinding,whichcauses

NAD(P)H-derived electrons to the heme (Figure 1). Dioxygen binds to the one electron-reduced ferrous hemeand thesecond electroncreatesthe ferricperoxy complex, which matures to the catalytically active ‘CompoundI’.Despiteaminoacidsequencedifferences ofupto90%,allP450sshareacommonfoldwithidentical topology and conserved secondary structural elements. Thequestionarises,howsuchahighlyconserved archi-tecture can sustain the observed immense variety in catalyzedreactions.Clearly,theP450foldevolvedearly asasafeplatformforaninherentlydangerousreaction– the activation of molecular oxygen– and as aversatile scaffold.Assuch,thevariabilityofP450reactionscannot beattributedtothecompositionandcapacityoftheactive site but israther aresultof theconcerted and dynamic action of the whole enzyme. A large body of research spanning both selective prokaryotic and highly promis-cuouseukaryoticP450sdemonstratestheessentialroleof plasticityintheselectionofsuitablesubstratesandtheir deliverytotheheme.

Questions concerning P450 flexibility involved in sub-strate binding have already been raised after the first crystal structure. In P450cam, the camphor substrate is effectivelysealedfromtheoutside,implyingastructural plasticitythatenablestheproteintoopenforsubstratesto enterandproductstoleave[39].Subsequentcrystaland NMR structures as wellas molecular dynamics simula-tions have since then confirmed how an impressive degreeofflexibilityinP450sfacilitatesastepwise adap-tationoftheenzymetothesubstrateinordertoleaditto theactive site.

BindingmechanismsinP450s

WorkonCYP3A4,ahumanP450involvedinxenobiotic metabolism, supportedaninduced fit substrate binding mechanism.Theenzymestructureincomplexwith mid-azolamhintsatsubstrate-induced,globalstructural read-justments,withconcurrentreshapingoftheactivesite.In particular,aconformationalswitchoftwohelices(theF– Gsegment)andlong-rangeresiduemovements transmit-ting from remote areas (the D, E, H, and I helices) triggered acollapse of theactive site cavity and ligand immobilization. Productive substrate positioning can occur attwo overlappingbinding sites near theIhelix, andasubstrateconcentration-dependentcollapseor wid-ening of the catalytic cavity determines the reaction’s regioselectivity[40].Structuralinvestigationsofthe pro-karyotic OleP in complex with a macrolactone are also consistentwithaninduced-fitbinding,wherebyacascade of interactionsresponsiblefor substrate-induced confor-mationalchangeswasproposed[41].SomeP450s, how-ever,wereshowntoexploreanincessantmotionbetween different conformations regardless of the presence of substrates.The ligand-freestructuresof the erythromy-cin-converting P450 EryK suggest the presence of a

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heterogeneous conformational ensemble between an openand aclosedstate[42].

Notably,theconformationalchangesoccurringupon sub-straterecognitioncanshow strikingsimilarities between verydistantrepresentatives.P450camandMycGareonly 29%identicalonsequencelevelandactonthestructurally diversesubstratescamphorandmycinamicinIV, respec-tively. Usingacombinationof NMRstructuralstudies, site-directedmutagenesisandfunctionalassays,severalregions farfromtheactivesiteofP450camweredemonstratedtobe criticaltoensureefficientrecognitionandorientationofthe substrate into the catalytic center. Many of the same secondarystructuralfeaturesinMycGareperturbedupon substratebinding.Themost-affectedresidueswere subse-quently found to be functionallyimportant and liein a conicalregionroughlyanti-symmetricwiththetriangular shapeoftheP450molecule[43].

P450s’substrateselectionviatailoredplasticity

Withtwelve entriesdepositedin theproteindatabank, CYP2B enzymes show one of the highest degree of plasticity among crystallographically characterized P450s — about one third of the protein is accounted forbyfiveplasticregions(PRs).ComparisonofPR2and PR4 allowed to distinguish fourdistinct conformations: ‘open’toallowsubstrateaccess,‘closed’and‘expanded’ upon binding of small and large ligands to CYP2B4, respectively, and an ‘intermediate’ form induced by and molded to the inhibitor 1-biphenyl-4-methyl-1H-imidazole(1-PBI)(Figure4a)[44].Ascatalysisinvolves

subtle, concerted conformational changes spanning a largepartoftheenzyme,allostericeffectsarefrequently observedandsometimesdrastic.InCYP2Bs,mutationsof residues remote from the active site caused not only a switch in selectivity for some substrates, but also pro-foundfunctionalchangesaffectingtheenzyme’scatalytic ratesandinhibition[45].Interestingly,mutations target-ingactive siteresidues producedmuchsmallerchanges [46].InCYP2B1,equallydistantmutationsenhancedthe metabolismofseveralsubstratesincludingtheanticancer prodrugs cyclophosphamide and ifosfamide [47]. Simi-larly, the enhanced activity of a rat CYP1A1 mutant toward adibenzo-p-dioxin toxin is triggered by a more efficientbindingofthesubstratein theactive siteeven though the mutation is over 25A˚ away [48]. In this scenario, it is not surprising how most of the single nucleotide polymorphisms (SNPs) that make CYP2B6 highlypolymorphicand,accordingly,differentlyactivein themetabolismofavarietyofdrugsliefarfromtheactive site of theenzyme[49]. Another demonstrationofhow the creation of a new activity passes mostly through mutationsinflexibleregionsinvolvedinsubstrate recog-nition [50] is the engineering of P450-BM3 toward a propane monooxygenase [51] where only a fraction of themutationswaslocatedin theactivesite(Figure 4b). TheroleofdynamicsoftheoverallP450foldisalsowell exemplified bythe long-range effects of putidaredoxin (Pdx) binding to the proximal face of P450cam, which influencesmotions onthe oppositeside of the protein. The open/close motion of the F/G helical region is

Figure4

(a) (b)

StructuralplasticityinP450s.(a)SuperimpositionoftheconformationsobservedforCYP2B.Theproteinisshownascartoonwithhelicesas cylinders.Regionsofconformationalvariabilityarehighlightedandcolouredwith‘open’(PDB1PO5,noligand),‘closed’(PDB1SUO,ligand: 4-CPI),‘expanded’(PDB2BDM,ligand:bifonazole),and‘intermediate’(PDB3G5N,ligand:1-PBI)inyellow,violet,green,andblue,respectively. Thehemecofactorisshownasredsticks.(b)TheP450-BM3hemedomainshownaswhite–bluecartoon,withthelocationsofthe

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contacts Pdx. The Pdx-induced changes in the F/G helicalregionareinstrumentaltocarryouttheenzymatic activity:ittriggersfreeanimportantaspartateinvolvedin the proton delivery network required for O2 activation

[52].Eventhe entranceof molecularoxygen intothe active site istunedbyproteindynamics.Simulationsof the protein backbone dynamics of P450-BM3 revealed thetransientnatureofsomechannels,withsubchannels forming and merging and O2 molecules hopping in

between[53,54].

The fullunderstanding of P450s catalysisis pivotal for exploiting their selectivity in industrial processes and designing tailored inhibitors for drug metabolism. The joint participationof remote,flexible elementscan rep-resentacomplication,astheirinfluenceonspecificityand catalyticactivitymaybedifficulttopredict.Thisexplains why directed evolution approaches with this enzyme family have been much more successful than rational approachesfocusedonactive-siteengineering.Apicture emergeswheretheactivesiteofP450s arereducedtoa mere accessoryrole.A recentstructuralcharacterization ofdifferentmembersofCYP153sillustratesthis.Among thesehomologs,allactivesiteresiduesareconserved,but theenzymesdisplayvaryinghydroxylationactivitieswith alkanes, fatty acids, and heterocyclic compounds. The comparison of fivecrystalstructures allowedto plotout the regions which exhibited the most pronounced sequence variabilities and conformational changes. In this manner, it was possible to identify the B/C-loop, theF,G,andHhelicesandtheF/G-looptobe responsi-blefor substraterecognitionandbinding[55].

Conclusions

While flavin-dependent MOs compensate their sub-domain’sintrinsicrigiditybylinkerandloopmovements and/orcofactormobility,P450scounterbalancetheheme cofactor’sinflexibilitybywidelydispersedmobileregions involvedinsubstratebinding.Thestructuraland mecha-nisticcomplexityfoundinflavoproteinMOsreflectsthe complex catalytic duty of efficiently coordinating three substrates bythesame active site in atimelyregulated fashion.Acompleteunderstandingofthereaction mech-anismreliesonfuturediscoveries,specificallywithregard to hydridetransferand substrateselectivitydifferences. WhenconsideringP450s,novelfeaturesoftheir mecha-nisms haveemergedfromvariousP450subfamilies.For both monooxygenase classes, it has become clear that structuraldynamicsplayanimportantrolein their cata-lytic functioning. Besides better understanding their molecularfunctioning,newinsightswillhopefullyclarify vast discrepancies in substrate acceptance and fuel the design of enzyme engineering strategies. Clearly, such rational approaches need to take all steps and loci involved in enzyme catalysis into consideration, rather

in astatic activesite.

Conflict

of

interest

statement

Nothingdeclared.

Acknowledgements

TheresearchforthisworkhasreceivedfundingfromtheEuropeanUnion (EU)projectROBOX(grantagreementno635734)underEU’sHorizon 2020ProgrammeResearchandInnovationactionsH2020-LEIT BIO-2014-1.

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Beyond active site residues: overall structural dynamics control catalysis in flavin-containing and heme-containing monooxygenases

University of Groningen