Schofield, 4PWC and4PWC 4PZF. are data available under database the Structural inPDB accession 4PVJ, 4PVE,4PVK, 4PVH, 4PWB, numbers Steric control dioxygenof reduction inflavoenzymes. Kingdom. Kingdom. This article is protected Allrights bycopyright. reserved. the VersionofRecord between thisversionand the copyediting, typesetting, pagination and proofreading process, which may to differences lead and for publication accepted This articlehasbeen Database: Running title: Article type: Original Article Corresponding address: ‡ 4 3 2 1 Domen Zafred, Austria; Email: [email protected]; Tel.: +43 699 1007 8283. GrazUniv Biochemistry, Zafred, of Institute Domen Dr. Present address: University Hospital Ghent, Ghent Un Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford, OX1 3TA, United andBiophysics, U.S.A. University, Oregon Biochemistry Oregon 97331, State of Department Univ Biosciences, Molecular of Institute Biochemistry,of UniversityInstitute Graz Accepted Article dioxygen reduction Rationally engineered flavin-dependent reveals steric control of 4 Silvia Silvia Wallner

1,2 BarbaraSteiner,

1 and Peter Macheroux Peter and

1 Andrea R. Teufelberger, ersity Graz,of A-8010 Graz,Austria. of Technology, A-8010 Graz, Austria. 1

. Please cite this article as doi: 10.1111/febs.13212 asdoi: . Pleasecitethisarticle undergone full peer review but has not been through buthasnotbeenthrough review undergone fullpeer ersity of Technology, Petersgasse 12, A-8010 Graz, iversity, De Pintelaan 185, 9000 Ghent, Belgium. 2‡ Altijana Hromic, 2 P. Andrew Karplus, Andrew P. 3 Christopher J. This article is protected Allrights bycopyright. reserved. be governed by a common fromoxidases othersuperfamilies revealed similar structur most important parameter affecting dioxygen reactivity in BBE-like . Analysis of flavin-dependent “” in the proximity of the flavin ring. We showed also that steric control of access to this site is the additional variants showed that our model enzymes have a cavity binding an anion and resembling the a its by factor reactivity (BBE) lowered dioxygen of 500. reactivity by a factor of 60,000. An inverse exchange (V alternate gatekeeper site (I153V) transformed the potential andredox highest activity the dehydrogenase dioxygen reactivity in the grass pollen allergen p Phl 4, amember thisof superfamily, whichhas glucose superfamily of flavoenzymes. We have identified an alternate gatekeeper residue that similarly controls residue was identified that controls the reactivity to can cause amino acid arapid()either sluggish(dehydrogenases) reaction. a Previously, or “gatekeeper” The ability of flavoenzymes to reduce dioxygen varies gr Abstract important role important aof positively chargedamino acidresidue ( oxidase fructosamine proximity of FAD in glucose oxidase ( potentials [5,9,1 flavin binding andaltered inhibitor been used identify to have Model flavoenzymes. this formation adduct of [12,13]. without intermediate in some flavin dependent oxidases [10,11], there isevidence that the reaction might also proceed characterizations singleof adductsatom suggested intermediate has never been observed in flavin dependent oxidases (Fig. 1) [5,9]. While the recent of electron transfer in reaction with dioxygen in flavoenzymes, however, the resulting hydroperoxide [7,8]. Based on the occurrence of the flavin-(4a)-adduct, th light dioxygenhave shed activation recent studiesof spreading itsrate sixover or relatively slowly with dioxygen, proteins flavin-bound ca appears to be the rate-limiting step in the process (Fig into ahydroxylated product, and the other into water. Kine hydrogen peroxide, or as monooxygenases, splitting the molecule such that one atom is incorporated to acceptor reducing as asan can function oxygen oxidases, dioxygen electron employ that Flavoenzymes [1–3]. reactions andcyclization formation complex halogenation, bond to amines, monooxygenation, Accepted Introduction Article In recent years several studies have insigh provided and [6] hasbeenmonooxygenases characterized adduct inFAD-dependent A flavin-(4a)-hydroperoxide reactions alcohols, and redox aldehydes, rangingof oxidations enzymes catalyze from dependent Flavin PDB ID: 3DJE mechanistic principle. mechanistic ders of magnitude [4,5]. [4,5]. of magnitude ders ) and monomeric oxidase ( oxidase sarcosine ) and monomeric PDB ID: 1GPE ID: PDB ) [15] or the conserved lysine in the proximity of N(5) in that the flavin-(4a)-hydroperoxy adduct is alikely dioxygen inproteinsfrom the vanillyl alcohol oxidase on the mechanism of dioxygen of the mechanism on . 1) and . 1) while free reduced insolution reactsflavin 169I) inthe169I) structurally related berberine bridgeenzyme Structural and biochemical characterization andbiochemical characterization of these and Structural measured in a flavoenzyme. A substitution at the eatly andiseatly controlled by theprotein environment that n decelerate or accelerate this reaction substantially, putative oxygen channels, influences of substrate of substrate influences and channels, oxygen putative to regulate the dioxygen reactivity of reduced flavins 4]. residues, such ashistidine H516 in the into an efficient oxidase by increasing dioxygen e reactive atom has been accepted asthe site al features suggesting thatdioxygen reactivity might tically,transfer the ofthe ts into the specific roles of certain residues in PDB ID: 1L9E utilization in utilization these enzymes first electron to dioxygen ) have suggested an position, suggesting that additional complexities exist oxidases listed in their study (see their supplementary Table 1) had an alanine or a proline at the gatekeeper increased oxygen reactivity 400-fold. The correlation in galactono- position suitable for the control of dioxygen reactivity in the VAO superfamily. In their model enzyme L- This atom. described what al.[23] carbon contains was motif Leferink residueet at astheby “gatekeeper” a “oxygen reactivity motif” and which creates the environm re towardstheactive sitecavity where substratebindingis the buried in the FAD binding domain and the redox-active isoalloxazine ring oriented with its structure with a have two-domain enzymes These cofactor bound [19,20]. bicovalently a or mono- employ which most structural oxidases and dehydrogenases, hydroxylases, FADof dependent includes superfamily) [PCMH] This article is protected Allrights bycopyright. reserved. cyclization of oxidative in cl06869) BBE California catalyzes poppy [24]. the from (superfamily thethe Database Conserved Domain subgro a are BBE-likeenzymes asmodels. p4 Phl allergen bridgeenzyme the berberine by employing superfamily the reactivity of the reduced flavin toward dioxygen. [16–18]. Despite this progress itis still questionable whet away to interact with the isoalloxazine ring or any residue ( attached at the flavin C6position) isin a loop above the covalently residue C166/C150 (the nitrogenof cysteine backbone (Fig. The p4,respectively 2A). and Phl near the isoalloxazine ring, such asH459/Y439 on the topology of the protein backbone are highly conserved, this is not the case for several amino acid side chains structural feature.novel finda to p sought and of Phl reactivity 4 we not suppressed oxygen al. explain et does by Leferink identified the gatekeeper position. Since this is afeature common to all known BBE-like enzymes [26], the gatekeeper vivo sluggishly with dioxygen suggesting that the FAD cofactor Phl BBE-like p4 Incontrast BBE enzymes, to characterized reacts reduced previously glucose. and other Despite these structural similarities, Phl p 4 does not catalyze C-Cbond formation, but instead oxidizes D- topologyoverall (RMSD of aligned C bridge [25]. Phl p 4 from Timothy grass shows extensive similarity to BBE (34% sequence identity), has asimilar BBE (Fig.2). This small cavity, with nearby partialposi cavity that ispositioned above the C(4a) carbon atom an carbonyl group. These two residues are oriented towards the to C(4)=O distance is hydrogenthe bond within W165/V149 preceding nitrogen theof backbone the In contrast, -face of the FAD is surrounded by aconserved 18-residue long segment of chain that we will refer to as the Accepted a having at despite an rather than a like oxidase, p dehydrogenase behaves words, Phl 4 . Inother Article In the present study, we have further investigated the control of oxygen reactivity in the VAO alcoholThe vanillyl oxidase (VAO)stru Closer inspection of the active sites of BBE and Phl γ -lactone dehydrogenase (GALDH), replacement of (GALDH), dehydrogenase replacement -lactone (S) -reticuline to α atoms is 1.53 Å) and aconserved bicovalent linkage of the FAD [22]. (S) -scoulerine by formation of a carbon-carbon bond, berberine -scoulerine a bond, formation the of carbon-carbon by ctural superfamily (also called the tively charged backbone nitrogens available for hydrogen available nitrogens backbone charged tively si in defining oxidase activity superfamily. oxidase inthe in defining (BBE, andthe EC pollen BBE-like protein 1.21.3.3) -faceH174/N158 or and V169/I153the on the VAO superfamily was not perfect asseven of the 30 organized by the substrate binding domain [21,22]. The The [21,22]. bindingdomain substrate bythe organized re d lined by the side chains of V169, H174 and H174 in V169, L145 side the chains of by d lined her there is acommon catalytic principle that governs is reoxidized by an alternative electron acceptor up of up of PCMH/VAO superfamily, classified aspfam08031 ent of the isoalloxazine ring above the C(4a) reactive -face of the flavin in BBE-like enzymes and too far p 4 revealed that while the position of FAD and the agatekeeper alanine with glycine (A113G) re -face ofthe flavin andplacedadjacent to a small e.g. by formation of a hydrogen bond, Fig. 2). bond, Fig.2). of formation ahydrogen by p -methylcresolhydroxylase re -faceBBE of si- face in much higher rates of reoxidation, with 7.1 and 3.9 x 10 a similar rate of reoxidation, however, both the single I153V and the I153V N158H double variant exhibited of 1.2 M was observed even at 10 mM glucose. In contrast to BBE, reduced wildtype Phl p 4 reacts with dioxygen ata rate (Table variant decreased 1). double further not inthe were measured the redox potentials of the various enzyme forms. The three BBE variants exhibited redox potentials VAOsuperfamily [25,30],of this andbecause the can playand other arole inreactivity, we (Table 1). this dehydrogenase into an oxidase by elevating its dioxygen reactivity more than four orders of magnitude a plays rolemajor in reactivitycontrolling dioxygen in This article is protected Allrights bycopyright. reserved. Biochemical characterization of an analogous oxygen pocket in the proximity of the reactive C(4a). families that appear to asimplications family, also have but flavoenzyme for have other we discuss,they enzyme like enzymes. These results not only make it possible to predict oxygen reactivity of members of the BBE-like identify this position as an alternate gatekeeper residue th variant had a remarkable 60,000-fold higher dioxygen reactivity compared to the wildtype enzyme, leading us to oxygen pocket formed by the oxygen reactivity motif on the variants (I153V, N158H and I153V N158H) to investigate the role of the side chains involved in shaping of the (Fig. 2B). cavity C not because inPhlthe p4 does exist pocket al.for aet Lindqvist similar describe feature structural oxygen inBBE-like oxidases. Inthispaper, we will call hole” in observed of the “oxyanion isreminiscent bonding, reoxidation of the reduced variants by dioxygen dropped by 500 to 1000-fold, from ~50,000 to ~50 M fold lower (V169I) rate constant for the reduction by its substrate and reductive rates isgiven in Table 1. In the case of BB andstorage [29]. during purification spirohydantoin slow double of formation indicated the variant N158H p single I153V Phl the 4 and I153V of Phl p 4 and Phl p 4 N158H were stable over longer periods of time, changes in the UV/Vis absorption spectrum pastoris Accepted Article Results ) homogeneity to andpurified asdescribed BBE in While variants, Procedures. Experimental wildtype Wildtype Wildtype p Phl andits4 three variants showed similar Bicovalent modification of the isoalloxazine ring contributes to the positive redox potential found inBBE redox potential positive the to ringcontributes isoalloxazine modification of the Bicovalent Based on these considerations, created considerations, Based these and aset we on BBE p Phl V169I) andG164A of V169I (G164A, 4 Kinetic parameters were determined for BBE as well as Phl p 4 variants and a summary of the oxidative All BBEand p4Phl variants we -1 s -1 , more than two orders of magnitude below that of free FAD (250 M re successfully expressed in the BBE and Phlp 4 variants δ methyl of group I153 projects towards theC(4a) andfillsthe it “oxygen pocket”, it“oxygen aterm that wasintroduced by Phl p4Phl andtheI153V variant very effectively converted d in glycolate oxidase [28]. Interestingly, the oxygen oxygen the [28]. Interestingly, oxidase d inglycolate 4 E, the single variants showed asimilar (G164A) and five- M -1 at is crucial for the control of oxygen reactivity in BBE- certain [27] and potentially able able bind to andpotentially [27] certain hydrolases s -1 re , respectively. Thus itis evident that residue I153 reactivity with glucose (Table 1) and no saturation -face of the isoalloxazine ring. The Phl p 4 I153V (S) Komagataella pastoris Komagataella -reticuline. In contrast, the rate of -1 s -1 ). The N158H variant showed (formerly Pichia -1 s -1 , and due to aclash with C structure with structure the p4highlights of wildtype Phl of this Å.Superposition 3.4 at approximately andV149 adistance C150 of nitrogens backboneof amide the by roughly 400 mV more positive than that of free FAD (Fig. 3,Table 1). its variants had redox potentials in the range of +200 mV, approaching the hadredox range approaching the variants O +200 potentialsits inthe mV, of the ion was fitted into the F (Table 2, Fig.4). The presence of the halide ion at the site was confirmed by an anomalous difference map and N158H p I153V andthe Phl 4 bromide for sodium being best results obtained the N158H), with andI153V (I153V experiments, an ion atlow occupancy was observed in the engineered oxygen pocket ofboth Phl p 4 variants surrogate for locating putative oxygen binding sites [31] Fig.4A). hasring (Table shown It for2, been flavoproteinsother that halideoxidases used ions can be asa isoalloxazine of the proximity inthe not but FAD binding of the domain, pockets placed inhydrophobic atoms the proximity of the reactive C(4a) carbon atom (Fig. 2). BBE and Phl p 4involved in control of oxygen reactivity concerns the existence of the oxygen pocket placed in cavity analogously to the wildtype Phl p 4. Thus it appears that the key structural difference between wildtype BBE V169I mutant was not determined, it isreasonable in direction of the ringslightly the the isoalloxazine andpushes FAD flavin). positioned analogously to the position of C(18) in 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin (Emmdh- adduct distal peroxy inour the atom was models (Fig.5).The four flavin derivatives of oxygen compared for what a sense in structures active sites canbe the modeled of p our and Phl 4 haveBBE variants, we carbon in the C(4a) position, but the shape of the bent isoa above the C(4a) atom on the This article is protected Allrights bycopyright. reserved. inall abutter adopted FAD p4structures our Phl sp to due bent Dioxygen reaction mechanism andputative transition states Structural features of the variants andcrystallographic experiments surrogateswith approximatelyof +110 mV, whichis in the gatekeeper position with an alanine, pocket similar to that in wildtype BBE. In the latter, th The I153V exchange in Phl p 4creates space at the andno structural respective were wildtype structure the observed. with perturbations superimposable virtually thatthechanges tuneproperties ofthe variants. Overa Phl p 4, we solved aset of crystal structures at 1.3 to 2.3 Åresolution (Table 2) to gain insight into the structural Acceptedthe reaction of reduced FAD with dioxygen is expected tobe further twisted due to sp Article

Xenon pressurization experiments with crystals of Intrigued by the strong effects of the V169I and G164A exchanges in BBE and the I153V substitution in 3 hybridization of the N5 and N10 atoms in the central pyrazine ring. The putative intermediate in δ atom of I153 (Fig.4B). o -F re c difference density. This anion bound in the oxygen pocket is positioned is ~2.5 positioned bound pocket Å This inthe density. oxygen anion difference -face, and~3.6 Åaway thefrom C comparablewildtype to enzyme(E i.e. G164A, where C where G164A, re -face theof isoalloxazine ring, resultinginformation of a e oxygen pocket is removed by replacement of the glycine ll, thestructures determined for p Phl 4 variants were to expect that the extra methyl group would fillthe and when halideions were usedin crystalour soaking fly bent shape [22], resembling reduced FAD is FAD reduced that resembling [22], shape fly bent that ion bindingisnot that feasib Phl p 4I153V yielded astructure with two xenon lloxazine ringderivative can significantly. vary To gain β of the alanine side chain protrudes towards the the towards alanineside chainprotrudes of the γ -methyl of V153 (Fig.4B). The is ion stabilized si -face (Fig. 2B). While the structure of the 0 = 132 mV, Table 1). Wildtype Phl p 4 and p 4 Phl Wildtype 1). Table mV, 132 = 2 /H 2 O 2 redox couple (270 mV), and mV), (270 couple redox 3 le inthe wildtype enzyme hybridization theof hybridization This article is protected Allrights bycopyright. reserved. for the control of dioxygen reactivity in the VAO superfamily. BBE andcharacteset of and was generated p4variants Phl variants did not show asubstantial effect on the rate of flavin reoxidation (Table 3). In the present study, a new N(10) on the inthe bondof proximity donors hydrogen the H459A, H174A and H104A BBE variants, the dioxygen flavoenzymes [9,17]. Although (atleast partial) positive studied inBBE implicated includepreviously residues corresponding with as important by other studies significant effect theon oxidative half reaction wi created to identify the residues involved in substrate binding and catalysis,none of these replacements had a BBE has been studied extensively and while several variants of amino acid exchange in the active site have been adduct at the at adduct derivatives are modeled in the structure of wildtype BBE (data not shown). When alignments with the peroxy Emmdh-flavin when except in allmodels Y439 of chain clashes but side the pocket C(4)=O into adduct fits with the oxygen N158H, p4I153V structure of Phl the residues in p4and residues Phl the oxidative half reaction of the reduced enzyme wi W165/V149 and C166/C150 and the ratesoxidative were caused exclusively by changes in causes steric hindranceisoleucine p inPhl conclude and 4 the We (Fig. 2 that 4). observed effects the on case of BBE, valine in this position allows access to p4Phl derivedalternate gatekeeper the C(4)=O where the gatekeeper resides at a conserved posi C(4a) (Fig. 4). This cavity can be removed by introduction reactivitymotif”two with backbone amidenitrogens pointingtowards the oxygen reactive and non-reactive variants is the existence of the “oxygen pocket” formed by the “oxygen ina dependent enzyme. flavin measured ever potential redox highest 1), proving that redox potential has no effect on oxygen reactivity even in Phl p 4, aprotein that exhibits the (Table the variants in any amino acidsubstitutions of substantially not bythe affected FADwere enzyme-bound reactivity isconsiderably lower than the reactivity of free reduced FAD (Table 1). The redox potentials of the (Table 1). Even though the decline in oxygen reactivity of the BBE variants was smaller, the attained level of stronger effect on dioxygen reactivity and introduction ofboth mutations did not further suppress the reaction oxygen reactivity (Table 1). Introduction of an alanine at aminoinverse acid exchange inBBEV169 gatekeeper” residue isoleucine I153 to valine prompted a60 Discussion Discussion group peroxy on protruding the with the of flavin-4a-hydroperoxides When models Accepted Article Reciprocal single amino acid substitutions at the X-ray crystal structure analysis enabled us to show that the only considerable difference between the si -face are performed, the C(4)=O atom fi vice versa (Table 1, Fig. 2 and Fig. 4). In the case re resides insome in VAO sub-families, -face of the FAD. The existence of the partial positive charge in the cavity I resulted in a lesspronounced butstill substantial 500-fold decrease in th dioxygen (Table 3) [25,29,32,33]. The substitutions the oxygen pocket whereas the th incorporating by dioxygen BBE-derivedamino acid the accessibility of the amide backbone nitrogens of isused (Fig. 6B). No clashes are observed when ts into the oxygen pocket (Fig. 6A). si charge in close proximity of the FAD was eliminated in the “gatekeeper” position of BBE of (G164A) hada position slightly the “gatekeeper” of asingle methyl group on either side: frontal above -face theof isoalloxazine ringinY106F andH459A BBE reactivity was hardly affected. Moreover, removing re rized with the aim to identify amino acids responsible -facetheFAD werecreated of to study the effecton ,000-fold increase in the rate of oxidation while the tion or on the opposite side of the cavity where the of Phl pofPhl4, substitution ofthe “alternate cluding theBBE-like In enzymes. the re -face of the FAD above the reactive additional methylgroup of re -face arealigned with oxygen pocket is on the In bindson the contrast organicthe substrate to the VAOsuperfamily, [10,39,40]. suchas cholin flavoenzymes well-studied other includes various proteases. PHBH is a member of the glucose–methanol–choline (GMC) family, which at resembles hole, the as atimeoxyanion feature of strongly already known akey bindingpocket the that They stressed backbone they nitrogens, bindingpocket”. bytwo oxygen formed which “carbonyl termed hole fits perfectly into the active site of realized [37]. that the [38] agostructure These Emmdh-flavin authors bySchreuder and coworkers decades of This article is protected Allrights bycopyright. reserved. Towards a unifying of dioxygen concept reactivity inflavoenzymes are present. and dehydrogenases Phl p 4 derived alternate gatekeeper position is either encoding BBE-like enzymes [26]), have an invariant glycine at the gatekeeper position, while the residue at the plant (the model plant kingdom inthe areabundant which enzymes, are possible within having sub-families members with so reactivity motif that moves the gatekeepers away from the gatekeeper position and isoleucine I110 at the alternate ga example isalditol oxidase (AldO, VAO there isno alternate gatekeeper residue that could amongconserved allVAO superfamily enzymes, the rest of in VAOsuperfamily enzymes. While the gatekeeper residue represent the dioxygen [5,35]. seen in previously the structure halidebindingsite and to VAO crystal was suggested similar (Fig. 7) was A sites [34,31]. not binding but reaction dioxygen at dioxygen sites, andthus pockets putative hydrophobic binds readily in atom xenon (Fig.4).Asshown cannot xenon inbefore, while pocket bound the oxygen can be a packed, ion halide thatthough even whichtightly showed experiments, soaking byour halide supported was features of FMN dependent glycolate oxidase oxidase ( glycolate FMN dependent of features was et studied al.who by described pocket” structuralLindqvist A few later years the acavity termed “oxygen (Fig. 8). reactivity was already discussed for flavoenzyme superfamilies. Interestingly, the existence and parameters structural inother exist equivalent whether we dioxygen reactivity, explored for essential 1FCB shift abackbone dueto ( isabsent proton donor in b2, the adehydrogenase which flavocytochrome structure glycolateof oxidase (Fig.8). Unlike inVAOand GMC where two amide groups serve as

Accepted This) [28]. pocket resides at the Article While universal predictions do not extend to all me Interestingly, the complete environment at the Since our study revealed that access to the oxygen pocket in flavoenzymes of the VAO superfamily is si -face, making thesefunctional similarities PDB ID: 2VFR PHBH with the C(4)=O gaining two strong hydrogen bonds upon moving into a p -hydroxybenzoate hydroxylase (PHBH, (PHBH, hydroxylase -hydroxybenzoate re -face ofthe andis filled FMN witha water molecule in the native ), which isan oxidase even thou PDB ID: 1GOX a valine, leucine or isoleucine, suggestingthat oxidases re e oxidase, glucose oxidase andD-amino acid oxidase lved representative structures. For example, BBE-like be alignedwith theI153 Anotherof p4Phl (Fig.7). -face of FADis defined by the oxygen reactivity motif tekeeper position. It is abackbone shift of the oxygen mbers theof VAOsuperfamily, reliablepredictions and position of the neighboring amide nitrogen are are nitrogen amide of the neighboring andposition the motif varies significantly (Fig. For instance,7). in reactive C(4a) in this enzyme (Table 4, Fig.7) [36]. role ananalogousof structural feature indioxygen and 1AL7 an interesting case convergentof evolution Arabidopsis thaliana Arabidopsis ). The protein was compared to PDB ID: 1PHH gh it has alanine A105 at the re -face of the flavin and the contains genes 28 contains ) more than two PDB ID: the oxygen pocket, occurring upon oxygen adduct formation at the ribityl chains a bound side moiety. in with strongly and histidine combination matrix and isseverely restricted in its movement in BBE-like proteins due to the covalent bonds to the cysteine in the active sites of our proteins with confidence, becaus observed in these derivatives, apparently due to the pr This article is protected Allrights bycopyright. reserved. Possible mechanisms of dioxygen reactivity backbone anda bond from ofT78 chain distance the glutamine (Fig.8).side Q127 oxygen of hydrogen a side byaserine at isformed chain positioned S106 oxidase glycolate of pocket oxygen donors, the proton the ring, however, in enzymes this isnot the case as the protein matrix renders the directly on the the on directly a play role indioxygenmajor reactivity in our model non-reactive variants. Therefore two scenarios are steric formation issterically possible on either side of alternate gatekeeper prevent movements of the carbonyl (Fig. 6).In the non-reactive variants alanine at the gatekeep acyl-CoA oxidase family exhibit two backbone nitrogen atoms at the where structural features analogous to the oxygen pocket have not been previously discussed. Enzymes of the (Fig. 5). Large variation of movements of carbonyl C(4)=O out of the plane upon C(4a) sp of Our found based inproteins structures three were flavin on derivatives, were (Fig. 1). models which four of with FAD derivatives, the putative reaction intermedia intermed andarelikely monooxygenases dependent in flavin reaction would be similar to the covalent derivatives. Si Nonetheless, we believe that conformation, orientation and allow us to distinguish aradical reaction from the reaction with ahydroperoxy intermediate (Fig.1). assume that oxygen reaction occurs at the C(4a) carbon in our model enzymes [4,9], but our experiments do not oxygen in its twisted conformation (Fig.6). [41]. In the case of fructosamine oxidase and monomeric C(4)=O, analogousfor to of the (Fig. proposed forPHBH movement that 8) upon hydrogen strong allow bonding Accepted Article protein matrix. the into ringis embedded isoalloxazine the how that a depends on inthe structural with great deal albeit of variation organization oxidases, flavoenzyme the pocket (Fig. 8). of information oxygen beinvolved nitrogen could of V105, backbone the assumed importance of the oxygen pocket, we suggest that this water molecule, together with the adjacent the active site removes astrongly bound water molecule in the proximity of the N(5) atom [17,18,42]. In light of We findsimilar structural patterns also in de flavin According to our modeling, major movements of the C(4)=O carbonyl group out of the plane and into Dioxygen reaction with free reduced flavin does not distinguish between the sides of the isoalloxazine isoalloxazine sidesof the the between does distinguish not reduced flavin reaction free with Dioxygen These examples illustrate that the re -face of FAD, or, ifdioxygen attack occurs from the concept an of “oxygen pocket” begenerallymight applicable to the ring in our oxidase variants, but only on the proteins; either theproteins; pocket thestabilizes peroxy adduct tes occurring during the proposed reoxidation reaction proposed reoxidation duringthe tes occurring otein environment. We were able to align these models nce C(4a) peroxy flavin derivatives have been observed observed been flavin have derivatives C(4a)peroxy nce ally possible where an accessible oxygen pocket could sarcosine oxidase, replacement of a conserved lysine in e the flavin ring is firmly anchored within the protein oxygen towards the proton donors. Similarly, adduct Similarly, adduct the donors. proton towards oxygen pendent oxidasesfrom othe er position as well as isoleucine in the position of the spatial arrangement radicalsof incase of the radical iates incertain oxidases [9 si si si -face, are only possible in oxidase variants -face, it stabilizes theC(4)=O carbonyl -face theof FAD, at a distance that would re - and r structural superfamilies si ], we have built built models ], have we 3 -face asymmetric. We hybridization can be hybridization si -face in the catalysis. of enzyme principle transition state isstabilized reoxidation duringthe of This article is protected Allrights bycopyright. reserved. Reagents Biotech. Biotech. proximity of the reactive C(4a) carbon atom. reactivity might be governed by similar principles, indicates superfamilies that dioxygen structures from other of ofthe oxidases flavin-dependent exploration alignment are within related subgroupssequence possible Furthermore, only highly of proteins. structurally reactivity of other members, however, reliable predicti suggests that acid amino replacements dioxygen reactivity in BBE-like enzymes. The conservati that structural these prediction demonstrated enable and of have the features we manipulation addition, reaction by controlling the access tothe oxygen pocket, structural results reveal that hydrophobic residues in the proximity to the isoalloxazine ring govern dioxygen analysis and Our a combined biochemical by site on active mutagenesis. residues design rational based of it isimportant to stress that both discussed reaction paths provide a rationale of how the sp enzymes of the acyl-CoA dehydrogenase or GMC superfamily, only one reaction mode is sterically feasible. Also, place incertain taking me be could both and theoretically Protein production was carried out inaBBI fermenter [43]Protein (Sartorius)CT5-2 asdescribed and stopped previously of the expression cassettes into the pastoris all from GenScript p Genes of and cloned pPICZ Phl into 4 were variants ordered used in ( p was study Phl this 4.0202 Non-glycosylated [44]. before a template for the polymerase chain reaction, the expression vector [pPICZ BBE G164A V169I] expression plasmids using the QuikChange® XL Site-Directed Mutagenesis Kit (Stratagene). As Cloni and U.S.A.) Louis, Experimental Procedures Conclusion

Acceptedng, expression and purification Article (formerly Chemicals were purchased and were Chemicals Sigma-Aldrich from In this study we have achieved substantial changes in the oxygen reactivity of flavin-dependent enzymes It isworth pointing out that the two reaction options in BBE-like enzymes may not be mutually exclusive Expression plasmids were transformed using electroporation into the expression strain expression strain the into usingelectroporation were plasmids transformed Expression Mutagenesis was performed to create the [pPICZ

(S) -reticuline was from the natural product collection of the Donald Danforth Plant Science Center (St. (rac) Pichia pastorisPichia -reticuline was synthesized asdescribed [43]. -reticuline was synthesized ) KM71H coexpressing the the coexpressing ) KM71H Komagataella with equivalent structural impact will havesimilar effects on the oxygen

genome was verified using colony polymerase chain reaction. i. e. the reduced flavinthe andthus a reduced rationalize fundamental the occurrence of and access to an oxygen pocket in the ons of dioxygen reactivity that are based solely on on of the oxygen pocket in the VAO/PCMH superfamily a feature that strongly resembles the oxyanion hole. In α mbers of the superfamily, whil - BBE V169I], [pPICZ -BBE V169I], oligonucleotide primers were ordered from VBC- from primers ordered were oligonucleotide S. cerevisiae UniProt Protein Database ID: B2ZWE9 protein disulfide . Integration α BBE-ER] was as used described α α -BBE and[pPICZ G164A], vector (Invitrogen). e insome cases, such as 3 hybridized hybridized Komagataella ). α - plotted Nernst [47]. according Nernst to equation plotted Minnaert dichlorophenolindophenol (DCPIP, E (DCPIP, dichlorophenolindophenol determination of the reductive rate constant respective substrate concentrations and fitting the resulting data with a non-linear hyperbolic curve led to to from 30 concentrations substrate 500 µM Hi-Tech). using diode(model followed array detector were cofactor aKinetaScanT MG-6560, andsubsequent inc flushingwith by nitrogen oxygen-free 25°C in an anaerobic atmosphere provided within agl KH amounts of of amounts the cofactor Reduction enzyme substoichiometric performedof solution. FAD with was substrate-reduced transients at 450 nm was performed with Ki This article is protected Allrights bycopyright. reserved. Crystallization, data collection Redox PotentialDetermination Transient Kinetics al.[45]. et from Nandy protocol byanadopted p4was of Phl purification done induction. Purification hof methanol of BBE performed and after was asdescribed100-150 [43] variants and ID23-1 (ESRF, Grenoble), and ID23-1 I04-1 crystal was frozen instantly afterpressure was released. Datasets were collected atbeam lines FIP-BM30A, ID29 bar will elsewhere). The andpressurized (details at be inahome-built of xenon described 40 device glycerol freezingto [34,31]. To obtain thestru with bromide, crystals were soaked composed of crystallization bufferwith 20% glycerol and fr (70% Tacsimate, pH 7.0), 43% PEG 2000 was used as the reservoir solution. Allcrystals were soaked in asolution in 20 mg/mL (7 protein drop mixing method, sitting the buffercrystallization (100 HEPES, pH7.5, mM ammo2.0 M Toluylene blue (E measured in 50 mM HEPES, pH 7.0.All experiments were carried out in aglove box as described above. with BBE variants were performed in 50 mM potassium ph 2 PO

Accepted Article4 , pH 6.7, with 100 mM NaCl, using 5 µM to 100 100 mM to 5 using µM NaCl, mM 100 with pH6.7, ,

Reductive half-reaction: Reductive at Hi-Tech) analyzed astopped-flow (SF-61DX2, half-reactions device were and with oxidative Reductive Oxidative half-reaction:

BBE G164A was crystallized inbatch, protein (3mixing °C at by determined from 25 an adapted potentials Olsonwere method etExperiments al.[46]. Redox (rac) -reticuline or glucose in order to prevent lag-phases in process. reoxidation lag-phases in the prevent to glucose order -reticuline or 0 =115 mV) was used as redox dye for redox potential determination BBE determination used for and of mV) asredox dye =115 was potential redox

Rates were determined by mixing air-saturated buffers with an oxygen free In BBE variants rate constants were determined in 100 mM Tris-HCl,pH 9.0 at (Diamond, (Diamond, (PetraDidcot) andP11 III,Hamburg). Data processed were with in crystallization solution with 20% glycerol and sodium bromide 3.5 M prior 0 =217 mV) for Phl p 4. The standard protein potentials were were potentials calculated from protein standard for The mV) Phl =217 p 4. cture with xenon, crystals were soaked and structural netic Studio Softwarenetic (TgK Scientific). Studio (S) k red -reticuline. Apparent rate constants were plotted against plotted against rate constantsthe were Apparent -reticuline. . Measurements of Phl p 4 variants were performed in 80 mM ove box(Belle Technology). Allsamples were rendered mM Tris pH mM 7.0) in3:2 ratio with crystallization buffer ubation in the glove box. Spectral changes of the flavin characterization osphate buffer, pH 7.0, while Phl p 4 while Phl buffer, were p4 pH7.0, variants osphate nium sulfate). p4Phl variants were crystallized using ozen inliquidnitrogen.ozen To final D-glucose concentrations. Fitting of obtained 5 5 in20 mg/mL Tris,pHmM 7.0) in1:1 ratio with in crystallizationsolution with20% obtain obtain the crystal structure 5 Mattevi be A(2006) orTo not to bean oxidase: the oxygen reactivitychallenging flavoenzymes. of This article is protected Allrights bycopyright. reserved. 4 Massey Activation of oxygen molecular V(1994) by and flavins . R, Miyanaga Stull2 F, Louie A, Teufel Michaudel Q, NoelJP, G, Baran PS, Palfey B&Moore BS RL&1 Fagan Palfey BA enzymes. Flavin-dependent (2010) In andenzymatically B.S.,A.H.expressed, BBE S.W., characterized purified S.W., D.Z., A.R.T. variants. proteins, characterized figures. prepared structurally Ma expressed, purifiedandenzymatically characterized Phl si and Coot [51]. Structures of wildtype Phl p 4 and variants have apositive electron density in the active site at the [48], refined andstructures wasconducted with with [50] were [49] Phenix replacement Phaser molecular XDS experiments. experiments. for their support andtechnical help with McDonough Jean-Luc Ferrer, andMichael von Delft Camattari, Frank andresourceful like for hisfinancialKeller We Drs.Andrea Walter support discussions. would thank to Zafred C.J.Schofield laboratory EMBO.of visit the to to ofD. grateful Prof. by We was are supported Prof. through SFBprojects F1805 and F4604 and by the PhD program “Molecular Enzymology” (W901). A short-term 3 Liu Y-C, Li Y-S, Lyu LiY-S, 3 LiuY-C, HsuL-J, S-Y, Chen Huang ChanH-C, Chou Chen HuangY-T, Y-H, C-J, C- G-H, well as figures were prepared with PyMol (Schroedinger) and finished with Gimp. [38]. Models as based adducts are structure of peroxy 4a,5-epoxyethano-3-methyl-4a,5-dihydrolumiflavin on the C(8) C(6), FAD and N(10). aligningatoms: in of Models by superimposed three were PyMol Proteins structures. indeposited it leave haveempty decided to impuritieswe the (like sulfite adduct) therefor andprotein glycerol -face theof FADthat varies insize References References Author contributions Acknowledgements Trends Biochem.Trends Sci. 22459–22462. (2013) Flavin-mediated dualan Flavin-mediated oxidation(2013) rearrangement. controls Favorskii-type enzymatic (Begley 7th ed), ed.,TP, 37–114. Elsevier, Amsterdam.pp. Accepted scaffolds. antimicrobial of Interception (2011) Li T-L & M-D Tsai C, 503 Article zurFörderung(FWF) “Fonds wissenschaftlichen the Forschung” wassupported research der This by

, 552–556.,

31 Nat. Chem. Biol. Nat. Chem. , 276–83., and shape.Itis probably derived from ingredients crystallizationof buffer,

7 , 304–9., teicoplanin oxidation intermediates yields newteicoplanin oxidation intermediates p 4 p 4 variants. designedD.Z. research, crystallized and nuscript nuscript S.W., P.M., by was C.J.S. P.A.K., D.Z., written Comprehensive Natural Products II J. Biol. Chem.

269 Nature ,

19 Leferink NGH, Heuts DPHM, Fraaije MW MW Heuts NGH, 19 Leferink & van Fraaije DPHM, Berkel (2008) VAO The flavoprotein WJH growing 18 Zhao BrucknerG, RC&Jorns MS (2008) Identification of the oxygen activation in site monomeric 16 Gadda G Oxygen(2012) activation in oxidases: theflavoprotein importance of positive. being JP 15 Klinman (2007) How doenzymes activate withoutoxygen inactivating themselves? A of human inthe12 Pennati oxidative an & half-reaction of intermediate Gadda Stabilization G (2011) 11 Spadiut O, TC, Tan Wongnate Krondorfer Sucharitakul Sygmund J, T, I, Haltrich D, C, Chaiyen P, S, Finnegan & RGadda Lountos 10 OrvilleG AM, GT, Prabhakar (2009) Crystallographic, 17 McDonald CA, Fagan RL, Collard F,17 McDonald RL, Monnier Fagan BA VM CA, &Palfey (2011) reactivityOxygen in This article is protected Allrights bycopyright. reserved. R, Riley14 Baron Chenprakhon P, C, A, RT, K, F, Thotsaporn Alfieri van Forneris Berkel Winter WJH, 13 Gannavaram S & Gadda Relative G(2013) timing of hydrogen and proton in transfers the reaction MW9 Chaiyen Fraaije P, &Mattevi A(2012) reaction ofenigmatic The withflavins oxygen. K, C4a- J, Chenprakhon8 P,Sucharitakul of MatteviThotsaporn Stabilization P(2011) A&Chaiyen Royant A, Pazmiño DE, M, MW C, Torres & Mattevi HM, R, Dudek 7 Orru A Martinoli Fraaije Weik 6 Vervoort J, Muller Lee F, J, van denBerg &Moonen WAM (1986) of CTW Identifications the true family. oxidase: sarcosine of Lys265role incatalysis. flavoenzymes: context matters. Biochemistry Res. oxidase. glycolate liver 8 rationalizes meleagris substrateAgaricus specificity andreveals a intermediate. flavin CK&Peterbauer Divne 1.6 C(2013) The Åcrystal of structure pyranose dehydrogenase from Biochemistry spectroscopic, andcomputational analysis a of diffusion into active diffusion flavoenzyme sites. MW, Chaiyen Fraaije P, MatteviMcCammon oxygen MultipleJA (2009) A & pathways guide oxidationof catalyzed byflavin cholineoxidase. Sci.Biochem. at N5 interactions flavin and C4aatoms. inatwo-component hydroperoxyflavin flavin-dependent monooxygenase is achieved through stabilization. catalysis: oxygen Snapshots of activation andintermediate (2011) enzymatic Baeyer-Villiger luciferase. nuclearcarbon-13 resonance magnetic spectrum of the inbacterial stable II intermediate

Accepted e53567., Article

40 Arch. Biochem.Arch. Biophys. , 325–33., Biochemistry

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286 , 29284–91.

J. Am. Chem. Soc. J. Am.Chem. 474 , 292–301.

50 Proc. Natl. Acad. Sci. U. S. A. Acad.Sci. Proc. Natl. , 1–3., J. Biol. J. Biol. Chem. Biochemistry flavin C4a-oxygen adductflavin incholineoxidase. C4a-oxygen Biochemistry

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106 , 10603–8. , Acc. Chem. PLoS One Trends

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284

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284 , D348–52., , 4392–7. FEBS J. Flavoproteins: 1 Biochemistry

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lent flavinylation in berberine bridge inberberine enzyme.lent flavinylation 50 (Hille R, Miller &Palfey SM, R, eds), pp.1–30.De (Hille B, , 5521–5534., Trends Biochem.Trends Sci. J. Biol.J. Chem. FEBS J.

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49 McCoy AJ, Grosse-Kunstleve Adams RW, PD, LC MD, Storoni &ReadRJ PhaserWinn (2007) 54 Bonivento D, Milczek EM, McDonald GR, Binda C, Holt A, Milczek BindaC, Edmondson McDonald EM, 54 Bonivento D, GR, DE &Mattevi A(2010) Lohkamp51 Emsley P, ScottB, & Cowtan Features K(2010) WG anddevelopment of Coot. Afonine50 Adams P PD, Bunkóczi V, ChenVB, G, Davis Echols IW, N, HeaddJJ, L-W, Hung Kapral (2010) XDS. Kabsch 48 W 47 Minnaert Measurement K(1965) of constant of thethe equilibrium reaction between cytochrome c This article is protected Allrights bycopyright. reserved. Table 1. Kinetic data PA& K 53 Karplus model Diederichs crystallographic and Linking (2012) data quality. 52 Mayhew (1999) SG The effects of pHand on semiquinone theformation oxidation-reduction 46 Stankovich Schopfer MT, LM &Massey of Determination V(1978) glucose oxidase oxidation- B 19 1. . 9 109 ± 2 110 ± 3 ±3 109 98 ± 9 ± 55 1 50 ± 3 ± 8 60 18.5 ± 1.4 0.04 ± 0.002 p4Phl wildtype ±13 106 BBE G164A V169I BBE V169I G164A BBE BBE wildtype Tables Tables crystallographic software. crystallographic 285 of ligand binding cooperativePotentiation through in effects monoamine oxidase B. 1030–3. Crystallogr. D. Biol. Crystallogr. system structurefor macromolecular solution. & aTC PH Zwart JS, comprehensive PHENIX: Richardson (2010) Terwilliger Python-based GJ, RW,Grosse-Kunstleve McCoy Moriarty R, AJ, NW, ReadRJ,Oeffner Richardson DC, and cytochrome a. Accepted of A mononucleotide. reappraisal. potentials flavin Article Chem. potentialsreduction and oxygenthe reactivity and of reduced fully semiquinoid forms. , 36849–56.,

253 a 0 (5± 1)·10 103 ± 4 , 4971–4979. Biochim. Biophys.Biochim. Acta and redoxpotentials. k red Acta Crystallogr. D. Biol. Crystallogr. D. Biol. Acta Crystallogr. [s -1 J. Appl. Crystallogr. d ] . . 211 ± 2 ± 0.1 1.3

66 , 486–501., k ox [M -1

110 s -1 4 ]E

Acta Crystallogr. D. Biol. Crystallogr. Acta D. Crystallogr. 40 , 42–56., , 658–674. Eur. J. Biochem. J.Eur. 132 ± 4 0 m]Oxygen reactivity motif [mV]

66 , 125–32. 147 AGVCPTVGVGGNFAGGGF 164 163 AAWCPTIGTGGHISGGGF 180 163 AGWCPTIGTGGHISGGGF180 163 AAWCPTVGTGGHISGGGF180 163 AGWCPTVGTGGHISGGGF180

265 , 698–702., Science

66 J. Chem. Biol. J. Biol. Biol. J. , 213–21. Acta Acta

336 ,

eudny 96(97 43(.) 96(89 1. 76 7. 7.) 94(12 8.3(8.2) 39.4(41.2) 78.6(77.2) 17.4(7.6) 19.6(18.9) 4.3(4.2) 19.6(19.7) Redundancy (%) Completeness reflections reflections Unique (outer shell) Data collection unit inasym. Mol. P6 Space group This article is protected Allrights bycopyright. reserved. 4PZF Resolution (Å) 4PWC Wavelength 4PWB 0.972 (Å) 0.915 0.92 4PVK 0.915 1.033 0.9800.92 4PVJ a/b/c (Å) 4PVH 4PVE Protein variant PDB ID Table 2. Data collection a d c b a Data taken from Mayhew [52]. Kinetic data taken from Winkler et al.[25]. Datataken from Massey [4]. O 53 ± 3 FADH Free 85 ± 1 108 ±Phl 1 p 4 I153V N158H Phl p 4 I153V Phl p 4 N158H k obs 2 /H at 1 mM glucose glucose 1 concentration. at mM 2

Accepted ArticleO 2 / / 270 270 / / 2 FD/ 250 /FAD / 0 10 9. 9.) 0 9.) 99.4( 100(99.9) 98.7(97.7) 100 (100) (12827) 130268 4 1 1 1 1 (1.554-1.5) 49.2-1.5 201.5 117.2/117.2/ wildtype Phl p 4 1 22 P6 2 (15565) 159566 (1.450-1.4) 40.7-1.4 201.1 118.0/118.0/ N158H Phl p 4 1 nd refinement statistics. 22 P6 2 d d (. ± (7.1 0.2)·10 (. ± (3.9 0.6)·10 d . . 208 ± 1 ± 0.4 2.1 63 75) 197673(18761) 76838 (7555) 201.7 117.7/117.7/ Phl p 4I153V (1.864-1.8) 49.4-1.8 1 22 P6 2 b (1.346-1.3) 49.3-1.3 1.0 117.4/117.4/20 N158H I153V Phl p 4 4 4 1 22 P6 2 57 10(0) 0 9.) 98.9(98.2) 100(99.9) 100 (100) 95.7) -209 -209 203 ± 1 205 ± 4 c c 54 65) 37013(3605) 65841 (6459) (1.968-1.9) 49.4-1.9 03.0 117.6/117.6/2 pressurized Xenon Phl p 4I153V 1 147 AGVCPTIGVGGHFAGGGF164 147 AGVCPTIGVGGNFAGGGF164 147 AGVCPTVGVGGHFAGGGF164 22 P6 2 (2.382-2.3) 29.3-2.3 1.5 117.2/117.2/20 3.5 MNaBr N158H I153V Phl p 4 1 22 P2 2

(13780) 140140 (2.279-2.2) 47.4-2.2 195.8 80.8/175.4/ G164A BBE 1

(%) (%) CC water (Å water This article is protected Allrights bycopyright. reserved. Table 3. Summarykinetic parameters of of BBEvariants. b a φ R Mean I/ R ae tm 2 70 0 61 7 10 308 150 377 601 (Å protein 508 Clash dev. (°) 700 angles Bond 3.38 score 5.11 1.91 dev. (Å) 1.78 2.01 1.39 length Bond 620 Water atoms Protein atoms 15713 3853 3851 3852 38543851 3853 φ Refinement CC Calculated with Phenix [50]. Phenix with Calculated Y106F Y106F H459A H174A C166A wildtype variant BBE H174N H174N H104A WORK SYM , , 2 ψ ψ 1/2 ) b b outliers outliers favoured () 01 18 005(.) .8(.) .9 22 03 54 03 31 0.12(1.1) 0.35(3.1) 0.38(5.4) 0.090(2.2) 0.28(2.9) 0.085(1.5) 0.12(1.8) (%) 1/2

a /R . 07) . 04) . 05) . 02) . 06) . 07) 1.0(0.79) 1.0(0.79) 1.0(0.69) 1.0(0.27) 1.0(0.54) 1.0(0.42) 1.0(0.76) is the correlation between two halves adataset, of as defined byKarplus andDiederichs [53]. σ FREE

Acceptedd Article a I 68(.) 18(.) 30(.) 85 07) 66(.) 49(.) 11.8(1.52) 24.9(2.0) 26.6(1.6) 18.58(0.71) 13.0(1.0) 11.8(1.1) 16.8(1.7) (I) c b d e 67±06 (3.2±0.3)·10 6.7±0.6 .8±00 (10±1)·10 0.28±0.02 .8±00 (7.0±0.3)·10 0.08±0.01 8 (2.9±0.3)·10 88±4 . . (8±1)·10 3.4±0.3 . . (4.1±1.7)·10 3.0±0.5 a .3/.5 .4/.6 .5/.8 .4/.6 010017 .8/.2 0.220/0.242 0.186/0.225 0.160/0.187 0.133/0.1540.146/0.1670.152/0.186 0.141/0.160

0 (5±1)·10 103±4 2 ) ) 44.2 46.3 35.1 34.1 33.4 35.0 32.5 041. 391. 2. 4. 54.1 48.5 24.4 20.4 17.823.9 19.5 0 0.5 0 0 0 0 95 96 96 96 96 .0 .0 .1 .0 13 008 0.004 0.008 1.34 0.009 0.011 0.008 .213 .313 00211 0.84 0.012 1.19 1.32 1.351.33 1.32 k red [s -1

] k ox [M -1 s -1 4 4 ] 0.54 ± 0.02 28 ± 4 28±4 132±4 0.54±0.02 8.0±0.2 k 4 0.48 ± 0.05 53 ± 2 53±2 0.48±0.05 4 4 3 4 0.7 ± 0.1 0.7±0.1 44±3 75±3 3.1±0.7 0.07±0.01 1.2±0.1 cat [s -1 ]

n.d. n.d. E 0 [mV] [mV] This article is protected Allrights bycopyright. reserved. gatekeeper. alternate the to analogous position spatial a at placed VAO the in residue isno * There the gatekeeper residues for various enzymes from the VAO superfamily. Table 4. Distances between the reactive C(4a) andthe closest side chain atom (or C e d c b a Kinetic datataken from Wallner et al. [33]. Kinetic datataken et from al.[25]. Winkler New datapublished in this paper. Kinetic datataken et from al.[29]. Winkler Kinetic datataken et from al.[32]. Winkler G164A G164A PDB ID 3D2H 4PZF 4PVE 4PVJ 1VAO 2VFR 2VFR 1VAO AldO 4PVJ VAO 4I153V p Phl 4PVE 4 p Phl (Å) 4PZF gatekeeper Alternate G164A BBE 3D2H dehydrogenas (Å) oxidase distance Gatekeeper, BBE Oxygen reactivity PDB ID Protein E417Q V169I G164A V169I G164A

Accepted Article e 18.5 ± 1.4 98 ± 9 0.02 ± 0.005 109 ± 3 109±3 0.02±0.005 98±9 18.5±1.4 d e 007±007 (5.3±0.2)·10 0.067±0.007 106 ± 13 60 ± 8 0.02 ± 0.002 109 ± 2 109±2 0.02±0.002 60±8 106±13 e 0.04 ± 0.002 50 ± 3 0.007 ± 0.001 110 ± 3 110±3 0.007±0.001 50±3 0.04±0.002 Val (4.9) Val (5.2) Val(5.2) Val (4.9) (4.7) Gly

Ala (3.7) Ala (3.7) eyrgns xds oiae oxidase oxidase e dehydrogenaseoxidase Gly (4.5) Gly (4.7) Pro (4.4) Ala (4.1) (4.1) Ala (4.4) Pro (4.7) Gly Ile (3.3) (4.5) Gly 4 0.054 ± 0.006 0.054±0.006 Val (4.7) N/A* Ile (5.1) Ile(5.1) N/A* (4.7) Val n.d. α ) of

intermediate found in monooxygenases has an in that found sp monooxygenases intermediate aradical pair(b)yields form pair [4]. The might First electron transfer in the reaction of a reduced FAD(a 1. Fig. [4]. formation abond without FAD (d).Altern oxidized yields peroxide hydrogen the This article is protected Allrights bycopyright. reserved. Accepted Article Oxidative half-reaction of Oxidative half-reaction flavoenyzme oxidases. a covalent bond to produce hydroperoxy adduct adduct (c), the produce hydroperoxy a covalent bond to

atively, the second electron transfer might take place 3 hybridized C(4a) atom, and subsequent elimination atom, elimination andof C(4a) subsequent hybridized ) with an oxygen molecule isthe rate limiting step that

[47]. Minnaert pl logarithmic double (B).The N158H p I153V Phl 4 mutant double (A) selected Phlp4 the andthe spectra offor course shown reduction are of of the Plots wildtype

(A) Aligned structures structures BBE(A) Aligned of wildtype (green, This article is protected Allrights bycopyright. reserved. binding V169/I153 & H174/N158 on the that correspond to weak interactions are in blue. The active sites are well conserved, with important exceptions: larger in andprotein matrix distances betweenare (C(6), flavin C(8) orange, bonds paper Hydrogen and N(10)). FAD (yellow). Three atoms from the isoalloxazine ring are used for the 3D alignment of proteins throughout this carbon atom (black). gatekeeper I169/I153 and the gatekeeper A164 extinguish ( G164A variant oxygen pocket together with the backbone nitrogen atoms of C166 and W165, the proposed proton donors. BBE Fig. 3. Fig. Accepted Article 2. Fig. si -face. (B)A view of the Redox potential determination. Redox potential BBE andPhlp 4 active sites.

PDB ID: 4PZF , purple) and Phl p 4 (pink) are aligned to show how side chains how show alternate to side arealigned of the and p4(pink) , purple) Phl re re -face of the isoalloxazine ring and H459/Y439 & E417/Q339 on the substrate substrate the on E417/Q339 & H459/Y439 and ring isoalloxazine the of -face -face of the FAD in wildtype BBE where a cavity (blue sphere)

PDB ID: 3D2H

the cavity by pointing towards the reactive C(4a) ots were used in the evaluation of data according to ) and p4( Phl PDB ID:4PVE , pink) inthe of proximity

forms the ring ( bi substrate The orientation. inred for easier H86 and the ( Phl p 4 inaligned ion andA164 isÅ wildtype Åand 1.5 structures, side the chains ofonly from our I153 the 1.7 nitrogen atoms of C150 and V149, the latter is in ahydrog (green mesh) ispositioned 2.5 Å above the reactive C(4a blue) accommodates abromide in the oxygen pocket. The oxygen surrogate placed into the difference omit map the FAD, is aligned from the bromide soak structure (Table 2). (B) Phl p 4 I153V N158H variant ( (A) Cartoon representation of Phl p 4 I153V variant ( This article is protected Allrights bycopyright. reserved. PDB ID: 4PVE Fig. 4. Fig. si

Accepted as ion, ingreen atoms shown at anda-face). presented black Xenon the are bromide spheres Article Structures with oxygen surrogates. , pink) andBBE ( G164A PDB4PZF ID: , purple), respectively. PDB ID: 4PWB ID: PDB ) (black atom in yellow FAD) and 3.4 Å FAD) and backbone ) (black in atom from 3.4 yellow nding siteis formed by alargecavity below the flavin en bond distance with the carbonyl O(4) (orange). In ) with FAD (yellow) in stick representation PDB ID:4PWC re -face of

,

oxidase B ( from protein X-ray structures: pyranose dehydrogenase ( pyranose dehydrogenase structures: X-ray protein from significantly. vary may FAD a from p4Phl structure is yellow, isbrown Emmdh-flavin andthree derivatives are of flavin derivatives shows that orientation of the adduct for placing the distal oxygen atom in a proposed orientat [38]. (Emmdh-flavin) The position4a,5-dihydrolumiflavin of position at which authors assigned an “unknown atom” [10]. (B) Drawing of the 4a,5-epoxyethano-3-methyl- derivative(A) Flavin from crystal structure of choline ( oxidase This article is protected Allrights bycopyright. reserved. Accepted Article 5. Fig. Four derivatives models usedofflavin putative states. for adduct C(4a) intermediate PDB ID: 2XCG ID: PDB , dark green) [54] andcholine oxidase ( and movement of the carbonyl C(4)=O out of the plane ion relative to the N(5). (C) 3D alignment of structures PDB4H7U ID: C(18) was used in our models of hydroperoxy flavins PDB2JBV ID: PDB ID: 2JBV , light green) [11], human monoamine human monoamine , green)[11], light ) with oxygen addu , cyan) [10]. ct placed at the

due to aclash with C( form in BBE G164A variant due to aclash with the gatekeeper, nor can it form in wildtype Phl p 4 or BBE V169I of nitrogens backbone the bonds (orange) with hydrogen adduct hydroperoxy C(4a) its on variants. Blue lines represent distances larger than 3.5 Å. shows that C(4)=O oxygen atom can freely move into the oxygen pocket in wildtype BBE and in Phl p 4 I153V on the derivative structure from choline oxidase ( based (A) residues. gatekeeper Model bythe and are aligned andBBE (purple) represented G164A p4(pink) Phl This article is protected Allrights bycopyright. reserved. blue) and position the bromide ion andposition isshown of itsblue) F with soak ( bromide N158H in structure p4I153V placed of Phl are the flavin derivative of Models Accepted Article 6. Fig. Models placedin putative intermediates of Phlp4. δ ) of the alternate gatekeeper residue I169/I153. re -face shows that the adduct fits into the oxygen pocket where itcan form PDB2JBV ID: o (B) Model based on the structure of Emmdh-flavin with with Emmdh-flavin of structure the on based (B) Model -F C150 and possibly also V149. This intermediate cannot c difference density (green). Structures of wildtype ) with C(4a) hydroperoxy adduct on its PDB ID:4PWC si -face

,

This article is protected Allrights bycopyright. reserved. bromide soak ( soak bromide p N158H structures I153V of Phl inaligned 4 inVAO isshown reactivity enzymes dioxygen defining The motif andnitrogen atoms. oxygen backbone bonds (shown hydrogen p two for in Phl andconserved strong with their4 through form N(3) and O(2) orange) that reside in the cavities of Phl p 4 and VAO, resp he pocket, therepresented andsize of position oxygen above O(4)) The highlyspheres rest of are conserved. is backbone lessthe influences the alignedwhich spatial the gatekeeper residues G148/P169/A105 and the position Fig. 7. Fig. Accepted Article The oxygenmotif. reactivity The PDB ID: 4PWC , blue), VAO, blue), ( PDB ID: 1VAO ID: PDB ectively. The motif ends in residues F164/V185/H121 which which F164/V185/H121 residues in ends motif The ectively. re by the bromide (brown) an , green) andAldO( the first donorof proton nitrogen backbone (blue

PDB ID: 2VFR ID: PDB d chloride (green)d chloride anions , purple). Position of of V105. backbone nitrogen andthe molecule be could the water pocket the oxygen donors of proton oxygen pocket in glycolate oxidase ( oxygen pocket at the acyl-CoA oxidase ( ( oxidase choline were mirror images. The putative oxygen pocket accommod At the first glance, environments of FADin Phl p 4 ( This article is protected Allrights bycopyright. reserved. crystal [28]. bound Astrongly water molecule isalso found infructosamine oxidase ( oxidoreductases isformed the by side chains S106of a Accepted 8. Fig. Article Flavoenzyme superfamilies. oxidases various structural from PDB ID: 2JBV PDB ID: 1IS2 si -face of the FAD where backbone nitrogens of P177 and G178 reside. The proposed proposed The reside. andG178 ofP177 nitrogens backbone FAD -face ofthe where , C) GMC like the to structural which, belongs superfamily. PHBH, Peroxysomal , D) belongs to the acyl-CoA dehydrogenase superfamily and has a putative andhasaputative superfamily , acyl-CoA dehydrogenase the to D)belongs PDB1GOX ID: , E) that belongs to the superfamily of FMN-linked PDB ID: 4PVE ID: PDB nd Q127 andisnd Q127 filled with a water in molecule the native ates C(4)=O carbonyl atom of FAD in the structure of , A)and ( PHBH PDB ID: 1PHH ID: PDB PDB ID:3DJE , B) look asif they , F) where the