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Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

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Oxidative Power: Tools for Assessing LPMO Activity on Cellulose biomolecules Review Oxidative Power: Tools for Assessing LPMO Activity on Cellulose Federica Calderaro 1,2,* , Loes E. Bevers 1 and Marco A. van den Berg 1 1 DSM Biotechnology Center, 2613 AX Delft, The Netherlands; [email protected] (L.E.B.); [email protected] (M.A.v.d.B.) 2 Molecular Enzymolog y Group, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands * Correspondence: [email protected]; Tel.: +31-6-36028569 Abstract: Lytic polysaccharide monooxygenases (LPMOs) have sparked a lot of research regarding their fascinating mode-of-action. Particularly, their boosting effect on top of the well-known cel- lulolytic enzymes in lignocellulosic hydrolysis makes them industrially relevant targets. As more characteristics of LPMO and its key role have been elucidated, the need for fast and reliable methods to assess its activity have become clear. Several aspects such as its co-substrates, electron donors, inhibiting factors, and the inhomogeneity of lignocellulose had to be considered during experimental design and data interpretation, as they can impact and often hamper outcomes. This review provides an overview of the currently available methods to measure LPMO activity, including their potential and limitations, and it is illustrated with practical examples. Keywords: lytic polysaccharide monooxygenase; enzyme assay; cellulose; lignocellulosic biomass; chromatography; enzyme kinetics Citation: Calderaro, F.; Bevers, L.E.; van den Berg, M.A. Oxidative Power: Tools for Assessing LPMO Activity 1. Introduction on Cellulose. Biomolecules 2021, 11, Until recently, the enzymatic conversion of recalcitrant polysaccharidic materials such 1098. https://doi.org/10.3390/ as chitin or cellulose was thought to solely depend on the activity of hydrolytic enzymes. biom11081098 The discovery of lytic polysaccharide monooxygenases (LPMOs) has revolutionized this view [1]. LPMOs are copper-containing enzymes that boost the activity of the classical Academic Editor: hydrolytic enzymes on either chitin [2] or cellulose [3]. In 2010, Vaaje-Kolstad et al. [1] Vijay Kumar Thakur showed that LPMO’s catalytic mechanism is based on an oxidative reaction, which re- sults in the cleavage of glycosidic bonds in polysaccharides, and an overall disruption of Received: 1 July 2021 the substrate’s structure, facilitating the activity of hydrolytic enzymes such as endoglu- Accepted: 22 July 2021 β Published: 26 July 2021 canase (EG), cellobiohydrolase (CBH), and -glucosidase (BG) (Figure1). LPMOs are now classified as auxiliary activities (AA) [4,5] and grouped into seven families in the CAZy database (CAZy. Available online: http://www.cazy.org/, accessed on 12 April 2021). Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in Enzymes belonging to families AA9 and AA10, originally known as GH61 and CBM33, published maps and institutional affil- respectively, were the first to be characterized [1,6]. AA9s include fungal enzymes that iations. are active on different substrates, such as cellulose [6–8], soluble cello-oligosaccharides [9], xylan [10], hemicelluloses [11–13], and starch [14,15]. AA10 LPMOs, found in all domains of life but mainly in viruses and bacteria, have been reported to have activity on chitin and cellulose [1,16]. AA11, AA13, and AA14 LPMOs are only found in fungi and show activity towards chitin, starch, and cellulose-bound xylan, respectively [14,17,18]. Mem- Copyright: © 2021 by the authors. bers of the AA15 family, found in Eukarya and viruses, show activity on both chitin and Licensee MDPI, Basel, Switzerland. This article is an open access article cellulose [19]. The most recently described family is AA16, with one enzyme so-far char- distributed under the terms and acterized showing activity on cellulose [20]. One peculiar structural feature of LPMO is conditions of the Creative Commons the presence of a flat binding surface in which two conserved histidines coordinate the 2+ Attribution (CC BY) license (https:// Cu ion and form the so-called histidine brace [6,21,22]. The third residue coordinating creativecommons.org/licenses/by/ the copper is a tyrosine for AA9 (Figure2), as well as for AA11/13/14/15 [ 18], and a 2+ + 4.0/). phenylalanine for most of the AA10 [23]. The reduction of Cu to Cu is driven by an Biomolecules 2021, 11, 1098. https://doi.org/10.3390/biom11081098 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, x FOR PEER REVIEW 2 of 21 Biomolecules 2021, 11, 1098 2 of 21 and a phenylalanine for most of the AA10 [23]. The reduction of Cu2+ to Cu+ is driven by an external electron donor, which can be a small molecule such as ascorbic acid or certain external electron donor, which can be a small molecule such as ascorbic acid or certain phenols [1,24,25], biomass components such as lignin and its derivatives [26,27], an en- phenols [1,24,25], biomass components such as lignin and its derivatives [26,27], an en- zyme such as cellobiose dehydrogenase and pyrroloquinoline quinone (PQQ)-dependent zyme such as cellobiose dehydrogenase and pyrroloquinoline quinone (PQQ)-dependent pyranose dehydrogenase (PDH) [28–33], or light [34–36]. This represents the first step in pyranose dehydrogenase (PDH) [28–33], or light [34–36]. This represents the first step in the oxidative reaction of LPMO. Following this, a reaction with O2 [1] or H2O2 [30] leads the oxidative reaction of LPMO. Following this, a reaction with O [1] or H O [30] leads to the hydroxylation of either the C1 (resulting in the formation of2 an aldonolactone)2 2 or to the hydroxylation of either the C1 (resulting in the formation of an aldonolactone) or C4 C4 carbon (with the formation of a 4-ketoaldose) of the glucose moieties in the polysac- carbon (with the formation of a 4-ketoaldose) of the glucose moieties in the polysaccharide charidesubstrate substrate [10,11], [10,11], releasing releasing both soluble both andsoluble insoluble and insoluble oxidized oxidized products products [6]. The majority[6]. The majorityof LPMOs of characterizedLPMOs characteriz so far,ed according so far, according to the CAZy to the database CAZy database (CAZy. Available (CAZy. Availa- online: blehttp://www.cazy.org/ online: http://www.cazy.org/, accessed, onaccessed12 April on 2021 12 April)[5], are2021 C1-) [5], or are C1/C4-oxidizing C1- or C1/C4- LPMOs,oxidiz- ingwith LPMOs, fewer strictlywith fewer C4-oxidizers strictly C4 reported-oxidizers in literature.reported in Over literature. the past Over few years,the past several few years,studies several have focusedstudies onhave the focused identification on the ofidentification elements involved of elements in LPMO involved regioselectivity in LPMO regioselectivitybecause the sequence-based because the sequence unambiguous-based prediction unambiguou of C1/C4-oxidizings prediction of C1/C4 activity-oxidizing is not as activitystraightforward. is not as straightforward. In this regard, the In precisethis regard, positioning the precise of the positioning active site of copper the active towards site copperthe substrate towards [37 the–40 substrate], which is [37 influenced–40], which by theis influenced local amino by acid the configuration,local amino acid appears config- to urationbe the main, appears determinant. to be the main determinant. Figure 1. Schematic representation of enzymes acting in the deconstruction of cellulose. LPMOs (C1- Figure 1. Schematic representation of enzymes acting in the deconstruction of cellulose. LPMOs (C1- oxidizing: EC 1.14.99.54; C4-oxidizing: EC1.14.99.56) use either O2 or H2O2 to hydrolyze cellulose oxidizing: EC 1.14.99.54; C4-oxidizing: EC1.14.99.56) use either O2 or H2O2 to hydrolyze cellulose chainschains in in the the crystalline crystalline regions, regions, making making the the cellulose cellulose more more accessible accessible for for the the action action of of hydrolytic hydrolytic enzymes.enzymes. Endoglucanases Endoglucanases (EG; (EG; EC EC 3.2.1.4) 3.2.1.4) hydrolyze hydrolyze the the glycosidic glycosidic bond bond in in the the amorphous amorphous regions regions ofof the the cellulose cellulose chains, chains, generating generating new new reducing reducing and and non non-reducing-reducing ends. ends. Cellobiohydrolases Cellobiohydrolases (EC (EC 3.2.1.91)3.2.1.91) processively processively release release cellobiose cellobiose units units from from the the reducing reducing (CBH (CBH I) I) and and non non-reducing-reducing (CBH (CBH II) II) endsends of of the the cellulose cellulose chains. chains. Cellobiose Cellobiose is is then then converted converted into into glucose glucose units units by by ββ-glucosidases-glucosidases (BG; (BG; ECEC 3.2.1.21). 3.2.1.21). Ox, Ox, oxidated oxidated glucose glucose moiety; moiety; CBM, CBM, carbohydrate carbohydrate binding binding module. module. Biomolecules 2021, 11Biomolecules, x FOR PEER2021 REVIEW, 11, 1098 3 of 21 3 of 21 Figure 2. CrystalFigure structure2. Crystal and structure catalytic and center catalytic of a fungalcenter of LPMO. a fungal Schematic LPMO. representationSchematic representation of the crystal of structurethe and a close-up viewcrystal of the structure histidine-brace and a close of- theup cellulose-activeview of the histidineTtLPMO9E-brace of from the Thielaviacellulose‑active terrestris Tt(UniProtLPMO9E ID from G2RGE5, PDB 3EJA, strictlyThielavia C1-oxidizing). terrestris (UniProt The copper ID atomG2RGE5, is shown PDB 3EJA, as an orangestrictly C1 sphere.-oxidizing). The residues The copper involved atom in is the shown coordination
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