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• W3100457323 abstract "Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides, thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose, but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall–derived hemicellulosic polysaccharides, and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharide degradation suggest that the subtle differences in amino-acid sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO subclusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)–linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries. Understanding enzymatic breakdown of plant biomass is crucial to develop nature-inspired biotechnological processes. Lytic polysaccharide monooxygenases (LPMOs) are microbial enzymes secreted by fungal saprotrophs involved in carbon recycling. LPMOs modify biomass by oxidatively cleaving polysaccharides, thereby enhancing the efficiency of glycoside hydrolases. Fungal AA9 LPMOs are active on cellulose, but some members also display activity on hemicelluloses and/or oligosaccharides. Although the active site subsites are well defined for a few model LPMOs, the molecular determinants driving broad substrate specificity are still not easily predictable. Based on bioinformatic clustering and sequence alignments, we selected seven fungal AA9 LPMOs that differ in the amino-acid residues constituting their subsites. Investigation of their substrate specificities revealed that all these LPMOs are active on cellulose and cello-oligosaccharides, as well as plant cell wall–derived hemicellulosic polysaccharides, and carry out C4 oxidative cleavage. The product profiles from cello-oligosaccharide degradation suggest that the subtle differences in amino-acid sequence within the substrate-binding loop regions lead to different preferred binding modes. Our functional analyses allowed us to probe the molecular determinants of substrate binding within two AA9 LPMO subclusters. Many wood-degrading fungal species rich in AA9 genes have at least one AA9 enzyme with structural loop features that allow recognition of short β-(1,4)–linked glucan chains. Time-course monitoring of these AA9 LPMOs on cello-oligosaccharides also provides a useful model system for mechanistic studies of LPMO catalysis. These results are valuable for the understanding of LPMO contribution to wood decaying process in nature and for the development of sustainable biorefineries. Efficient conversion of plant biomass for the production of biofuels and other sustainable bioproducts and materials is considered largely dependent on the key enzymes lytic polysaccharide monooxygenases (LPMOs) (1Johansen K.S. Lytic polysaccharide monooxygenases: the microbial power tool for lignocellulose degradation.Trends Plant. Sci. 2016; 21: 926-936Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 2Tandrup T. Frandsen K.E.H. Johansen K.S. Berrin J.-G. Lo Leggio L. Recent insights into lytic polysaccharide monooxygenases (LPMOs).Biochem. Soc. Trans. 2018; 46: 1431-1447Crossref PubMed Scopus (39) Google Scholar). LPMOs are copper-dependent oxidoreductases that use molecular oxygen or hydrogen peroxide as cosubstrate to cleave polysaccharides (e.g. cellulose, hemicellulose, chitin, starch) through an oxidative mechanism (3Vaaje-Kolstad G. Westereng B. Horn S.J. Liu Z. Zhai H. Sorlie M. Eijsink V.G. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides.Science. 2010; 330: 219-222Crossref PubMed Scopus (714) Google Scholar, 4Bissaro B. Rohr A.K. Muller G. Chylenski P. Skaugen M. Forsberg Z. Horn S.J. Vaaje-Kolstad G. Eijsink V.G.H. Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2.Nat. Chem. Biol. 2017; 13: 1123-1128Crossref PubMed Scopus (199) Google Scholar, 5Walton P.H. Davies G.J. On the catalytic mechanisms of lytic polysaccharide monooxygenases.Curr. Opin. Chem. Biol. 2016; 31: 195-207Crossref PubMed Scopus (132) Google Scholar). LPMOs exist either as single-domain enzymes or appended to other protein domains, for example, carbohydrate-binding modules (CBMs) (6Lenfant N. Hainaut M. Terrapon N. Drula E. Lombard V. Henrissat B. A bioinformatics analysis of 3400 lytic polysaccharide oxidases from family AA9.Carbohydr. Res. 2017; 448: 166-174Crossref PubMed Scopus (31) Google Scholar, 7Levasseur A. Drula E. Lombard V. Coutinho P.M. Henrissat B. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes.Biotechnol. Biofuels. 2013; 6: 41Crossref PubMed Scopus (639) Google Scholar), directing the enzymes to their target substrate (8Crouch L.I. Labourel A. Walton P.H. Davies G.J. Gilbert H.J. The contribution of non-catalytic carbohydrate binding modules to the activity of lytic polysaccharide monooxygenases.J. Biol. Chem. 2016; 291: 7439-7449Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, 9Courtade G. Forsberg Z. Heggset E.B. Eijsink V.G.H. Aachmann F.L. The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation.J. Biol. Chem. 2018; 293: 13006-13015Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 10Chalak A. Villares A. Moreau C. Haon M. Grisel S. d'Orlando A. Herpoël-Gimbert I. Labourel A. Cathala B. Berrin J.-G. Influence of the carbohydrate-binding module on the activity of a fungal AA9 lytic polysaccharide monooxygenase on cellulosic substrates.Biotechnol. Biofuels. 2019; 12: 206Crossref PubMed Scopus (18) Google Scholar). To date, LPMOs are grouped into seven families in the CAZy database (11Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42: D490-D495Crossref PubMed Scopus (3271) Google Scholar), namely the auxiliary activity (AA) families AA9-AA11 and AA13-AA16 (7Levasseur A. Drula E. Lombard V. Coutinho P.M. Henrissat B. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes.Biotechnol. Biofuels. 2013; 6: 41Crossref PubMed Scopus (639) Google Scholar), in which members have been found mainly in bacteria and filamentous fungi (12Filiatrault-Chastel C. Navarro D. Haon M. Grisel S. Herpoel-Gimbert I. Chevret D. Fanuel M. Henrissat B. Heiss-Blanquet S. Margeot A. Berrin J.G. AA16, a new lytic polysaccharide monooxygenase family identified in fungal secretomes.Biotechnol. Biofuels. 2019; 12: 55Crossref PubMed Scopus (62) Google Scholar, 13Couturier M. Ladeveze S. Sulzenbacher G. Ciano L. Fanuel M. Moreau C. Villares A. Cathala B. Chaspoul F. Frandsen K.E. Labourel A. Herpoel-Gimbert I. Grisel S. Haon M. Lenfant N. et al.Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.Nat. Chem. Biol. 2018; 14: 306-310Crossref PubMed Scopus (136) Google Scholar, 14Hemsworth G.R. Henrissat B. Davies G.J. Walton P.H. Discovery and characterization of a new family of lytic polysaccharide monooxygenases.Nat. Chem. Biol. 2014; 10: 122-126Crossref PubMed Scopus (225) Google Scholar, 15Lo Leggio L. Simmons T.J. Poulsen J.-C.N. Frandsen K.E.H. Hemsworth G.R. Stringer M.A. von Freiesleben P. Tovborg M. Johansen K.S. De Maria L. Harris P.V. Soong C.-L. Dupree P. Tryfona T. Lenfant N. et al.Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.Nat. Commun. 2015; 6: 5961Crossref PubMed Scopus (173) Google Scholar, 16Vu V.V. Beeson W.T. Span E.A. Farquhar E.R. Marletta M.A. A family of starch-active polysaccharide monooxygenases.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 13822-13827Crossref PubMed Scopus (155) Google Scholar, 17Harris P.V. Welner D. McFarland K.C. Re E. Navarro Poulsen J.C. Brown K. Salbo R. Ding H. Vlasenko E. Merino S. Xu F. Cherry J. Larsen S. Lo Leggio L. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.Biochemistry. 2010; 49: 3305-3316Crossref PubMed Scopus (527) Google Scholar), but also recently in arthropod and fern species (18Sabbadin F. Hemsworth G.R. Ciano L. Henrissat B. Dupree P. Tryfona T. Marques R.D.S. Sweeney S.T. Besser K. Elias L. Pesante G. Li Y. Dowle A.A. Bates R. Gomez L.D. et al.An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.Nat. Commun. 2018; 9: 756Crossref PubMed Scopus (107) Google Scholar, 19Yadav S.K. Archana Singh R. Singh P.K. Vasudev P.G. Insecticidal fern protein Tma12 is possibly a lytic polysaccharide monooxygenase.Planta. 2019; 249: 1987-1996Crossref PubMed Scopus (20) Google Scholar). In all LPMOs studied to date, the active-site redox center is made up of the strictly conserved histidine brace (His-brace) motif, which consists of the N-terminal His and a second His involved in the coordination of one copper atom (20Quinlan R.J. Sweeney M.D. Lo Leggio L. Otten H. Poulsen J.C.N. Johansen K.S. Krogh K.B.R.M. Jorgensen C.I. Tovborg M. Anthonsen A. Tryfona T. Walter C.P. Dupree P. Xu F. Davies G.J. et al.Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 15079-15084Crossref PubMed Scopus (579) Google Scholar, 21Frandsen K.E.H. Lo Leggio L. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes.IUCrJ. 2016; 3: 448-467Crossref PubMed Scopus (45) Google Scholar). The AA9 family also contains some members without any LPMO activity, lacking both the His-brace and copper, while features implicated in carbohydrate binding have been maintained (22Frandsen K.E.H. Tovborg M. Jorgensen C.I. Spodsberg N. Rosso M.N. Hemsworth G.R. Garman E.F. Grime G.W. Poulsen J.C.N. Batth T.S. Miyauchi S. Lipzen A. Daum C. Grigoriev I.V. Johansen K.S. et al.Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.J. Biol. Chem. 2019; 294: 17117-17130Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar). Genes encoding AA9 LPMO enzymes are found in high numbers in fungal saprotrophs. For instance, species of the Lentinus, Phanerochaete, Neurospora, and Podospora genera display between 14 and 33 AA9 genes (https://mycocosm.jgi.doe.gov/mycocosm/home); however, the reason for this gene multiplicity is unknown. Structurally, these AA9 isoforms share a central β-sandwich core, but the surface-exposed structural features around the His-brace vary as reviewed (2Tandrup T. Frandsen K.E.H. Johansen K.S. Berrin J.-G. Lo Leggio L. Recent insights into lytic polysaccharide monooxygenases (LPMOs).Biochem. Soc. Trans. 2018; 46: 1431-1447Crossref PubMed Scopus (39) Google Scholar, 21Frandsen K.E.H. Lo Leggio L. Lytic polysaccharide monooxygenases: a crystallographer's view on a new class of biomass-degrading enzymes.IUCrJ. 2016; 3: 448-467Crossref PubMed Scopus (45) Google Scholar, 23Vaaje-Kolstad G. Forsberg Z. Loose J.S.M. Bissaro B. Eijsink V.G.H. Structural diversity of lytic polysaccharide monooxygenases.Curr. Opin. Struct. Biol. 2017; 44: 67-76Crossref PubMed Scopus (89) Google Scholar). The LPMO structure from Thermoascus aurantiacus (TaAA9A) (20Quinlan R.J. Sweeney M.D. Lo Leggio L. Otten H. Poulsen J.C.N. Johansen K.S. Krogh K.B.R.M. Jorgensen C.I. Tovborg M. Anthonsen A. Tryfona T. Walter C.P. Dupree P. Xu F. Davies G.J. et al.Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components.Proc. Natl. Acad. Sci. U. S. A. 2011; 108: 15079-15084Crossref PubMed Scopus (579) Google Scholar) revealed a Cu-bound His-brace on a flat extended surface near a conserved Tyr, in the C-terminal (LC) loop, putatively involved in substrate binding. AA9 LPMOs were initially assumed to specifically target crystalline regions of polysaccharides, but later, the activity was also demonstrated on a range of hemicellulose polysaccharides such as xyloglucan (XG), xylan, glucomannan (GM), and mixed-linkage β-glucans (MLGs), and also on oligosaccharides (17Harris P.V. Welner D. McFarland K.C. Re E. Navarro Poulsen J.C. Brown K. Salbo R. Ding H. Vlasenko E. Merino S. Xu F. Cherry J. Larsen S. Lo Leggio L. Stimulation of lignocellulosic biomass hydrolysis by proteins of glycoside hydrolase family 61: structure and function of a large, enigmatic family.Biochemistry. 2010; 49: 3305-3316Crossref PubMed Scopus (527) Google Scholar, 24Agger J.W. Isaksen T. Varnai A. Vidal-Melgosa S. Willats W.G. Ludwig R. Horn S.J. Eijsink V.G. Westereng B. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6287-6292Crossref PubMed Scopus (233) Google Scholar, 25Isaksen T. Westereng B. Aachmann F.L. Agger J.W. Kracher D. Kittl R. Ludwig R. Haltrich D. Eijsink V.G. Horn S.J. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.J. Biol. Chem. 2014; 289: 2632-2642Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 26Huttner S. Varnai A. Petrovic D.M. Bach C.X. Kim Anh D.T. Thanh V.N. Eijsink V.G.H. Larsbrink J. Olsson L. Specific xylan activity revealed for AA9 lytic polysaccharide monooxygenases of the thermophilic fungus Malbranchea cinnamomea by functional characterization.Appl. Environ. Microbiol. 2019; 85: 1408-1419Crossref Scopus (18) Google Scholar, 27Frandsen K.E. Simmons T.J. Dupree P. Poulsen J.C. Hemsworth G.R. Ciano L. Johnston E.M. Tovborg M. Johansen K.S. von Freiesleben P. Marmuse L. Fort S. Cottaz S. Driguez H. Henrissat B. et al.The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.Nat. Chem. Biol. 2016; 12: 298-303Crossref PubMed Scopus (169) Google Scholar, 28Simmons T.J. Frandsen K.E.H. Ciano L. Tryfona T. Lenfant N. Poulsen J.C. Wilson L.F.L. Tandrup T. Tovborg M. Schnorr K. Johansen K.S. Henrissat B. Walton P.H. Lo Leggio L. Dupree P. Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.Nat. Commun. 2017; 8: 1064Crossref PubMed Scopus (75) Google Scholar, 29Bennati-Granier C. Garajova S. Champion C. Grisel S. Haon M. Zhou S. Fanuel M. Ropartz D. Rogniaux H. Gimbert I. Record E. Berrin J.G. Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.Biotechnol. Biofuels. 2015; 8: 90Crossref PubMed Scopus (144) Google Scholar, 30Fanuel M. Garajova S. Ropartz D. McGregor N. Brumer H. Rogniaux H. Berrin J.-G. The Podospora anserina lytic polysaccharide monooxygenase PaLPMO9H catalyzes oxidative cleavage of diverse plant cell wall matrix glycans.Biotechnol. Biofuels. 2017; 10: 63Crossref PubMed Scopus (31) Google Scholar, 31Basotra N. Dhiman S.S. Agrawal D. Sani R.K. Tsang A. Chadha B.S. Characterization of a novel lytic polysaccharide monooxygenase from Malbranchea cinnamomea exhibiting dual catalytic behavior.Carbohydr. Res. 2019; 478: 46-53Crossref PubMed Scopus (14) Google Scholar). The AA9 LPMO from Neurospora crassa (NcAA9C) is active on both cello-oligosaccharides and hemicellulosic substrates, suggesting that the enzyme recognizes small stretches of β-1,4-linked glucosyl units (24Agger J.W. Isaksen T. Varnai A. Vidal-Melgosa S. Willats W.G. Ludwig R. Horn S.J. Eijsink V.G. Westereng B. Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 6287-6292Crossref PubMed Scopus (233) Google Scholar, 25Isaksen T. Westereng B. Aachmann F.L. Agger J.W. Kracher D. Kittl R. Ludwig R. Haltrich D. Eijsink V.G. Horn S.J. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.J. Biol. Chem. 2014; 289: 2632-2642Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). Borisova et al. (32Borisova A.S. Isaksen T. Dimarogona M. Kognole A.A. Mathiesen G. Varnai A. Rohr A.K. Payne C.M. Sorlie M. Sandgren M. Eijsink V.G. Structural and functional characterization of a lytic polysaccharide monooxygenase with broad substrate specificity.J. Biol. Chem. 2015; 290: 22955-22969Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) determined the structure of NcAA9C and noticed three consecutive Asn residues in the L2 loop and an insertion in the L3 loop of NcAA9C structure compared with other AA9 LPMOs (such as TaAA9A) acting on insoluble cellulosic substrates (Fig. 1 and Fig. S1). Around the same time, we found that an AA9 LPMO from Podospora anserina (PaAA9H) with an L3 loop similar to that of NcAA9C showed activity on cello-oligosaccharides as well as XG and GM (29Bennati-Granier C. Garajova S. Champion C. Grisel S. Haon M. Zhou S. Fanuel M. Ropartz D. Rogniaux H. Gimbert I. Record E. Berrin J.G. Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by Podospora anserina.Biotechnol. Biofuels. 2015; 8: 90Crossref PubMed Scopus (144) Google Scholar). Shortly after, we determined the structures of two AA9 LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A, previously Chaetomium virescens), which both belong to the phylogenetic cluster C38 (see Results) and display activity on cello-oligosaccharides and on XG, MLG, and GM (28Simmons T.J. Frandsen K.E.H. Ciano L. Tryfona T. Lenfant N. Poulsen J.C. Wilson L.F.L. Tandrup T. Tovborg M. Schnorr K. Johansen K.S. Henrissat B. Walton P.H. Lo Leggio L. Dupree P. Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.Nat. Commun. 2017; 8: 1064Crossref PubMed Scopus (75) Google Scholar, 33Tandrup T. Tryfona T. Frandsen K.E.H. Johansen K.S. Dupree P. Lo Leggio L. Oligosaccharide binding and thermostability of two related aa9 lytic polysaccharide monooxygenases.Biochemistry. 2020; 59: 3347-3358Crossref PubMed Scopus (5) Google Scholar). The L3 loops in LsAA9A and CvAA9A are slightly shorter than those in NcAA9C and PaAA9H; however, their L3 loops are all extended compared with those of TaAA9A, which is not active on oligosaccharides. Interestingly, truncation of the L3 loop in NcAA9C abolishes XG activity (34Laurent C. Sun P. Scheiblbrandner S. Csarman F. Cannazza P. Frommhagen M. van Berkel W.J.H. Oostenbrink C. Kabel M.A. Ludwig R. Influence of lytic polysaccharide monooxygenase active site segments on activity and affinity.Int. J. Mol. Sci. 2019; 20: 6219Crossref Scopus (16) Google Scholar), and for a closely related (about 60% sequence identity) AA9 LPMO from Chaetomium thermophilum (denoted as CtPMO1 [35Chen C. Chen J. Geng Z. Wang M. Liu N. Li D. Regioselectivity of oxidation by a polysaccharide monooxygenase from Chaetomium thermophilum.Biotechnol. Biofuels. 2018; 11: 155Crossref PubMed Scopus (15) Google Scholar]), a His-to-Ala mutation in the L3 loop (equivalent to His66 in LsAA9A, see below) has been shown to abolish celloheptaose activity. The first LPMO–carbohydrate complex structures determined were LsAA9A in complex with cello-oligosaccharides (27Frandsen K.E. Simmons T.J. Dupree P. Poulsen J.C. Hemsworth G.R. Ciano L. Johnston E.M. Tovborg M. Johansen K.S. von Freiesleben P. Marmuse L. Fort S. Cottaz S. Driguez H. Henrissat B. et al.The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.Nat. Chem. Biol. 2016; 12: 298-303Crossref PubMed Scopus (169) Google Scholar, 36Frandsen K.E.H. Poulsen J.-C.N. Tandrup T. Lo Leggio L. Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH.Carbohydr. Res. 2017; 448: 187-190Crossref PubMed Scopus (16) Google Scholar). Later, LsAA9A was determined in complex with oligosaccharides derived from hemicelluloses (xylan, GM, and MLG) (28Simmons T.J. Frandsen K.E.H. Ciano L. Tryfona T. Lenfant N. Poulsen J.C. Wilson L.F.L. Tandrup T. Tovborg M. Schnorr K. Johansen K.S. Henrissat B. Walton P.H. Lo Leggio L. Dupree P. Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.Nat. Commun. 2017; 8: 1064Crossref PubMed Scopus (75) Google Scholar). The LsAA9 complex structures confirmed that the oligosaccharides were bound by one Asn in the L2 loop, polar amino-acid residues in the L3 loop, and the conserved Tyr platform (Tyr 203) in the LC loop but also via charged residues in the L8 loop (after notation [37Span E.A. Suess D.L.M. Deller M.C. Britt R.D. Marletta M.A. The role of the secondary coordination sphere in a fungal polysaccharide monooxygenase.ACS Chem. Biol. 2017; 12: 1095-1103Crossref PubMed Scopus (57) Google Scholar]). Subsites from −4 to +2 were defined based on the LsAA9–cellohexaose (cell6) complex. The negative subsites (−4 to −1) were formed by the platform Tyr203 in the LC loop as well as Glu148, Asp150, and Arg159 in the L8 loop, whereas the positive subsites (+1 to +2) were formed by Asn28 in the L2 loop and His66 and Asn67 in the L3 loop. Interestingly, the structures also revealed an uncommon lone pair—π aromatic interaction between the pyranose ring O5 and the imidazole of the N-terminal His of the His brace (27Frandsen K.E. Simmons T.J. Dupree P. Poulsen J.C. Hemsworth G.R. Ciano L. Johnston E.M. Tovborg M. Johansen K.S. von Freiesleben P. Marmuse L. Fort S. Cottaz S. Driguez H. Henrissat B. et al.The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.Nat. Chem. Biol. 2016; 12: 298-303Crossref PubMed Scopus (169) Google Scholar, 36Frandsen K.E.H. Poulsen J.-C.N. Tandrup T. Lo Leggio L. Unliganded and substrate bound structures of the cellooligosaccharide active lytic polysaccharide monooxygenase LsAA9A at low pH.Carbohydr. Res. 2017; 448: 187-190Crossref PubMed Scopus (16) Google Scholar). Very recently, crystallographic cello-oligosaccharide complexes were also obtained with CvAA9A and showed broadly the same interactions as LsAA9A (Tandrup et al, submitted; 6YDC, 6YDD, and 6YDE) (33Tandrup T. Tryfona T. Frandsen K.E.H. Johansen K.S. Dupree P. Lo Leggio L. Oligosaccharide binding and thermostability of two related aa9 lytic polysaccharide monooxygenases.Biochemistry. 2020; 59: 3347-3358Crossref PubMed Scopus (5) Google Scholar). Although the substrate-binding determinants and active-site subsites are well defined for LsAA9A, the molecular determinants of substrate specificity are still not easily predictable for other AA9 LPMOs. In this study, we focused on seven LsAA9A-related enzymes from fungal saprotrophs to provide further insights into the main molecular determinants driving LPMO activity toward cello-oligosaccharides and hemicelluloses. The selection of AA9 sequences was carried out bioinformatically by searching for sequences with at least 50% sequence identity to LsAA9A in the CAZy (http://www.cazy.org/) and JGI Mycocosm (https://mycocosm.jgi.doe.gov/mycocosm/home) databases. We retrieved 100 sequences mainly from basidiomycete species belonging to the Agaricomycetes class but also from ascomycete species belonging to the Gyromitra and Aspergillus genera (Pezizomycetes or Eurotiomycetes class, respectively). The vast majority of these species hold around 20 or more AA9 LPMO genes. Most of these fungi appeared only once in the list, whereas some appeared twice (Gyromitra esculenta, Armillaria ostoyae, Crepidotus variabilis, Botryobasidium botryosum, Schizophyllum commune, Volvariella volvacea) or even three or four times (Crucibulum laeve and Coprinellus pellucidus). It means that these fungi display at least one LsAA9A-related LPMO that may have a dedicated biological function. LsAA9A belongs to C38, and the 100 LsAA9A-related sequences are classified within either subcluster C29 or C38, as previously defined by Lenfant et al. (6Lenfant N. Hainaut M. Terrapon N. Drula E. Lombard V. Henrissat B. A bioinformatics analysis of 3400 lytic polysaccharide oxidases from family AA9.Carbohydr. Res. 2017; 448: 166-174Crossref PubMed Scopus (31) Google Scholar) based on the sequence conservation of AA9 N-terminal halves, that is, the N-terminal part of the sequences bordered by the residues forming the His brace. Of the 100 sequences, seven sequences were finally selected based on the composition of their putative substrate-interacting loops, L2, L3, L8, and LC (Fig. 1 and Fig. S1). The five AA9 sequences belonging to subcluster C38 originate from the basidiomycetes Phanerochaete chrysosporium (PchAA9E JGI protein ID 2934397), Phanerochaete carnosa (PcaAA9A 261285), Bjerkandera adusta (BaAA9A 353490), Armillaria gallica (AgAA9A 500811), and Schizophyllum commune (ScAA9A 2617723) and share between 63 and 76% sequence identity with LsAA9A (Table 1). Two other AA9s belong to subcluster C29 and originate from the ascomycetes Aspergillus oryzae (AoAA9A UniProt Q2US83) and Aspergillus fumigatus (AfAA9C GenBank EAL85444.1) (Fig. 1, Tables 1, and S1). AoAA9A and AfAA9C naturally harbor a CBM1 module, and their catalytic AA9 domains share 53 and 55% sequence identity with LsAA9A, respectively (Table 1). Some of the candidates chosen in this study originate from well-known fungal species for which other AA9 LPMOs have already been characterized, for example, P. chrysosporium PcGH61D (38Wu M. Beckham G.T. Larsson A.M. Ishida T. Kim S. Payne C.M. Himmel M.E. Crowley M.F. Horn S.J. Westereng B. Igarashi K. Samejima M. Stahlberg J. Eijsink V.G. Sandgren M. Crystal structure and computational characterization of the lytic polysaccharide monooxygenase GH61D from the Basidiomycota fungus Phanerochaete chrysosporium.J. Biol. Chem. 2013; 288: 12828-12839Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar, 39Westereng B. Ishida T. Vaaje-Kolstad G. Wu M. Eijsink V.G. Igarashi K. Samejima M. Stahlberg J. Horn S.J. Sandgren M. The putative endoglucanase PcGH61D from Phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose.PLoS One. 2011; 6e27807Crossref PubMed Scopus (188) Google Scholar) and A. fumigatus AfAA9B (40Lo Leggio L. Weihe C.D. Poulsen J.-C.N. Sweeney M. Rasmussen F. Lin J. De Maria L. Wogulis M. Structure of a lytic polysaccharide monooxygenase from Aspergillus fumigatus and an engineered thermostable variant.Carbohydr. Res. 2018; 469: 55-59Crossref PubMed Scopus (19) Google Scholar). However, no activity on oligosaccharides or hemicelluloses has been reported for any AA9 enzymes from these organisms.Table 1LsAA9A homologue sequencesEnzyme nameOrganismAA9 subclusterSequence identity to LsAA9ASubstitutionsNegative subsitesPositive subsitesLsAA9ALentinus similisC38-n/an/aPchAA9EPhanerochaete chrysosporiumC3876%E148QPcaAA9APhanerochaete carnosaC3874%E148QBaAA9ABjerkandera adustaC3869%E148QN67DAgAA9AArmillaria gallicaC3864%D150Y, R159KN67DScAA9ASchizophyllum communeC3863%Y203WN67DAoAA9AAspergillus oryzaeC2955%D150N, S151R, R159KN67DAfAA9CAspergillus fumigatusC2953%D150N, S151R, R159KN67DSubcluster 38 (C38), subcluster 29 (C29), negative subsites (−4, −3, −2, and −1), positive subsites (+1 and +2).For each AA9 enzyme name, the organism of origin and the cluster they belong to are given. The sequences are listed by their identity to LsAA9A and the substitutions affecting the subsite on either side of the scissile bond are indicated under substitutions. Open table in a new tab Subcluster 38 (C38), subcluster 29 (C29), negative subsites (−4, −3, −2, and −1), positive subsites (+1 and +2). For each AA9 enzyme name, the organism of origin and the cluster they belong to are given. The sequences are listed by their identity to LsAA9A and the substitutions affecting the subsite on either side of the scissile bond are indicated under substitutions. Compared with LsAA9A, the sequences of PchAA9, PcaAA9, and BaAA9 have a conservative substitution of the substrate-interacting Glu148 to a Gln near the −2 subsite, which we will refer to as E148Q. From here on, we will use this notation to refer to natural variations of the selected AA9 LPMOs compared with LsAA9A (note that this notation does not refer to mutant variants). The sequence of BaAA9 has an additional N67D substitution potentially affecting the +2 subsite. The sequences of AgAA9, ScAA9A, AoAA9, and AfAA9 also have the N67D substitution together with other ones near the negative subsites. ScAA9A has an Y203W substitution of the otherwise almost completely conserved platform Tyr involved in stacking interactions with the glycosyl unit in subsite −3. AgAA9A has a D150Y substitution, and because the Asp in LsAA9A interact" @default.
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• W3100457323 title "Identification of the molecular determinants driving the substrate specificity of fungal lytic polysaccharide monooxygenases (LPMOs)" @default.
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