Two routes of oxidative xyloglucan degradation by lytic polysaccharide monooxygenases from Neurospora crassa
Peicheng Sun , Christophe V.F.P. Laurent , Matthias Frommhagen , Willem J.H. van Berkel , Roland Ludwig  and Mirjam A. Kabel (email@example.com) 
 Laboratory of Food Chemistry, Wageningen University & Research, Bornse Weilanden 9, 6708 WG Wageningen, The Netherlands
 Vienna Institute of BioTechnology, BOKU-University of Natural Resources and Life Sciences, 1190 Vienna, Austria
Fungi boost the deconstruction of lignocellulosic plant biomass via oxidation using lytic polysaccharide monooxygenases (LPMOs). LPMOs act in synergy with hydrolytic carbohydrate degrading enzymes and are classified in the CAZy database (CAZy.org; ) in Auxiliary Activities (AA) 9-11 and 13-16. Although all so far characterised AA9 LPMOs show oxidative cleavage of cellulose, only a few AA9 members were identified to also oxidatively cleave hemicelluloses, in particular xyloglucan . Furthermore, detailed xyloglucan cleavage patterns and structures of (oxidised) products are poorly understood, mainly due to a lack of standards and suboptimal analytical methods available.
Hence, our study aimed to determine the different cleavage patterns of two xyloglucan-active AA9 LPMOs from Neurospora crassa (NcLPMOs) by characterising the oxidative xyloglucan degradation products with various analytical approaches. Hereto, xyloglucan degradation products formed by the previously described NcLPMO9C  and by a newly discovered xyloglucan-active NcLPMO9M were investigated. Using HPAEC-PAD and MALDI-TOF MS, we confirmed that NcLPMO9C cleaved tamarind seed xyloglucan (TXG) via C4-oxidation, while NcLPMO9M showed oxidative cleavage of TXG resulting in both C1- and C4-oxidised products.
Further analysis using HILIC-MS, revealed that NcLPMO9C specifically cleaved TXG next to single unsubstituted glucosyl units, resulting in C4-oxidised unsubstituted glucosyl terminal residues. Interestingly, NcLPMO9M cleaved TXG non-specifically next to both substituted and unsubstituted glucosyl units, even next to glucosyl units substituted by the xylosyl-galactosyl side-chain. NcLPMO9M was also determined to cleave the black currant xyloglucan next to glucosyl units having xylosyl-galactosyl-fucosyl side-chains. Our study provides a detailed understanding of two different routes of LPMO-xyloglucan degradation and is a contribution to envision future applications of LPMOs within the degradation and modification of xyloglucan.
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