ARTICLE https://doi.org/10.1038/s41467-020-19561-8 OPEN Kinetic insights into the peroxygenase activity of cellulose-active lytic polysaccharide monooxygenases (LPMOs) ✉ Riin Kont1, Bastien Bissaro 2,3, Vincent G. H. Eijsink 2 & Priit Väljamäe 1 Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in Nature, where they catalyze the hydroxylation of glycosidic bonds in polysaccharides. Despite the importance of 1234567890():,; LPMOs in the global carbon cycle and in industrial biomass conversion, the catalytic prop- erties of these monocopper enzymes remain enigmatic. Strikingly, there is a remarkable lack of kinetic data, likely due to a multitude of experimental challenges related to the insoluble nature of LPMO substrates, like cellulose and chitin, and to the occurrence of multiple side reactions. Here, we employed competition between well characterized reference enzymes and LPMOs for the H2O2 co-substrate to kinetically characterize LPMO-catalyzed cellulose oxidation. LPMOs of both bacterial and fungal origin showed high peroxygenase efficiencies, 5 6 −1 −1 with kcat/KmH2O2 values in the order of 10 –10 M s . Besides providing crucial insight into the cellulolytic peroxygenase reaction, these results show that LPMOs belonging to multiple families and active on multiple substrates are true peroxygenases. 1 Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia. 2 Faculty of Chemistry, Biotechnology and Food Science, NMBU—Norwegian University of Life Sciences, Ås, Norway. 3 INRAE, Aix Marseille University, UMR1163 Biodiversité et Biotechnologie Fongiques, 13009 Marseille, France. ✉ email: [email protected] NATURE COMMUNICATIONS | (2020) 11:5786 | https://doi.org/10.1038/s41467-020-19561-8 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19561-8 ytic polysaccharide monooxygenases (LPMOs) are mono- is a non-heme-iron peroxidase rather than an oxidase, we have Lcopper enzymes involved in degradation of polysaccharides, developed a theoretical framework and an experimental setup including cellulose and chitin, which are the most abundant to kinetically characterize a so far non-characterized and very polysaccharides in Nature. Their oxidative mechanism was dis- important LPMO reaction, the cellulolytic peroxygenase reac- covered in 2010 by showing that the chitin-binding protein tion. We have addressed this issue by setting up competition CBP211,2 of the bacterium Serratia marcescens (here referred to as experiments with a kinetically well characterized reference SmAA10A) catalyzes oxidative cleavage of β-1,4 glycosidic bonds enzyme and an LPMO of interest, to derive k /K values cat mH2O2 in chitin, while generating C1-oxidized oligosaccharide products3. for LPMOs acting on soluble cellooligosaccharides as well as The reaction was shown to be dependent on the presence of O2 insoluble cellulose (Avicel). One of the two reference enzymes and reductant3. Ten years of intensive research has revealed the in this study was a chitin-active bacterial LPMO, SmAA10A, for – presence of LPMOs in most kingdoms of life3 7 and today, these which detailed kinetic data exist thanks to the availability of enzymes are classified within seven families of auxiliary activities unique, but not generally available, experimental tools that (AA)8 in the database of carbohydrate-active enzymes9, with include the use of 14C-labeled chitin nanowhiskers (CNWs)14. families AA9, from fungal origin, and AA10, primarily from The other reference enzyme was readily available horseradish bacterial origin, being the best studied. To date, many LPMOs, peroxidase (HRP) acting on two different, both readily avail- acting on various oligo- and polysaccharides, and using different able, substrates, N-acetyl-3,7-dihydroxyphenoxazine (Amplex regioselectivities of oxidation, have been identified10. Red, AR) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic While structurally and functionally well characterized7,11, kinetic acid) diammonium salt (ABTS). The cellulose-active LPMOs studies of LPMOs are scarce. The scarcity of kinetic data likely studied were fungal family AA9 LPMOs from Trichoderma reflects the numerous experimental challenges associated with reesei (TrAA9A) and Neurospora crassa (NcAA9C), as well as a quantitative characterization of LPMO functionality12.Thecom- bacterial AA10 LPMO from Streptomyces coelicolor (ScAA10C, plexity is exemplified by the recent discovery that LPMOs may use formerly also known as CelS2). 13–20 H2O2, rather than, or even instead of, O2, as co-substrate . Regardless of the mechanism, LPMOs need an external electron donor for catalysis. In the H2O2-driven reaction, the reductant is Results used only in a “priming-reduction” of the Cu(II) resting state to the Two enzymes competing for the same co-substrate—kinetic 13,18,21 catalytically active Cu(I) form whereas in the O2-driven foundation. Let us consider a system where two enzymes, the reaction the reductant is consumed in stoichiometric amounts (i.e., reference enzyme and the enzyme of interest compete for the 10,22 delivery of two electrons per glycosidic bond cleavage) .The H2O2 co-substrate (Fig. 1). The substrate of the reference enzyme auto oxidation of reductants by O2 often leads to the formation of is designated as Sref and that of the enzyme of interest as S. The H2O2 and this complicates the interpretation of kinetic data. Fur- experimental approach used in this study requires that the values thermore, LPMOs that are free from substrate have both reductant of the kinetic parameters of the reference enzyme are known. To oxidase23 and peroxidase16,18 activity where the oxidase activity this end, we used the well-characterized chitin-active SmAA10A 17,23–25 leads to the formation of H2O2 . as well as HRP as reference enzymes (see below). To date only two detailed kinetic studies of LPMO action on Enzyme reactions involving two substrates may follow a carbohydrate substrates are available. Kuusk et al.14 have shown ternary complex (both substrates are bound to the enzyme) or a that the H2O2-driven oxidation of insoluble chitin by (bacterial) ping-pong mechanism. Depending on the order of the binding of 6 −1 −1 SmAA10A has a kcat/KmH O in the order of 10 M s ,whereas substrates the mechanisms involving a ternary complex are 26 2 2 Hangasky et al. have shown that O2-driven oxidation of soluble further grouped as random order or ordered mechanisms. In the cellohexaosebyanLPMOfromthefungusMyceliophthora ther- absence of products, the steady-state rates of H2O2 consumption mophilia (MtPMO9E) has a k /K in the order of 103 M−1 s−1. cat mO2 (d[H2O2]/dt) for an enzyme obeying an ordered (with S being the While the relative importance of the O2-andH2O2-driven reaction first substrate to bind) ternary complex or a ping-pong mechanisms remains a subject of debate15, available data clearly show that the peroxygenase reaction is much faster14,18,22,27. Unfortunately, due to a lack of suitable methods, so far, there are no ref ref Measurement S S -O Interest solid kinetic data for one of the most important LPMO reactions, V = f ([LP MO ]) namely oxidative degradation of insoluble cellulose. ref 27 28–30 In Nature as well as in industrial applications , LPMOs Eref involved in lignocellulose degradation operate in redox-active (Sm AA10A; HRP) ref environments where both potential co-substrates, O2 and H2O2,as kcat H2O2 Competition H2O ref well as multiple compounds and/or enzymes interacting with Km these co-substrates, are present. For example, the secretomes of Interest fungi growing on lignocellulosic biomass contain, next to LPMOs, LPMO a myriad of different enzymes including several H2O2-consuming 27 Kinetic parameters enzymes like different peroxidases and peroxygenases . Com- deduction parative studies of the k /K values of the various H O - S S-O cat mH2O2 2 2 consuming enzymes in such secretomes could provide some Fig. 1 Two enzymes, a reference enzyme (Eref, i.e., SmAA10A or HRP) insight into the relevance of the H2O2-driven LPMO reaction and fl and an LPMO of interest compete for the H O co-substrate. The rate of the ow of H2O2 through different enzyme reactions. In this 2 2 the reference reaction (v ) is a function of the concentration of the LPMO regard, one must bear in mind that the use of kcat/K values as ref mH2O2 k K performance metrics in comparing different enzymes assumes that of interest. Provided with the cat/ m (for H2O2) value for reference reaction one can calculate the kcat/Km (for H2O2) value for the LPMO the concentration of H2O2 is much lower than the apparent K values of the competing enzymes31. reaction (oxidation of substrate, S). Note that, in the case of HRP, the mH2O2 ref Here, inspired by the work of Wang et al.32 who demon- reference substrate (S ) is oxidized but the oxygen atom is not ref strated in 2013 that the fosfomycin-producing epoxidase HppE incorporated into S . 2 NATURE COMMUNICATIONS | (2020) 11:5786 | https://doi.org/10.1038/s41467-020-19561-8 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-020-19561-8 ARTICLE 33,34 mechanism are given by Eqs. (1) and (2), respectively . absence of competing enzyme). Provided with the value of [E]50 ½ and the values of the parameters of the reference reaction, the d H2O2 v ¼À apparent kcat/K for the enzyme of interest can be calculated ref dt mH2O2 Âà Âà using Eq. (6). kref Eref ½H O Sref ! ! ! ¼ cat 2 2 Âà ÂÃ; app app Âà ref ref ref ref ref ref ref ref K K þ K ½þH O K S þ ½H O S kcat kcat E iS mH2O2 mS 2 2 mH2O2 2 2 ¼ : ð6Þ ref ½ KmH O K E ð1Þ 2 2 mH2O2 50 Âà Âà Finally, the true kcat/K value can be found from the ½ ref ref ½ref mH2O2 d H2O2 kcat E HÂÃ2O2 S Âà analysis of (k /K )app values measured at different [S], v ¼À ¼ : cat mH2O2 ref ref ½þref ref þ ½ref dt KmS H2O2 KmH O S H2O2 S according to Eq.