Abstract
Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity, and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate-active redox enzymes.
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References
Vaaje-Kolstad G, Westereng B, Horn SJ, Liu ZL, Zhai H, Sorlie M, Eijsink VGH (2010) An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science 330(6001):219–222. doi:10.1126/science.1192231
Loose JS, Forsberg Z, Fraaije MW, Eijsink VG, Vaaje-Kolstad G (2014) A rapid quantitative activity assay shows that the Vibrio cholerae colonization factor GbpA is an active lytic polysaccharide monooxygenase. FEBS Lett 588(18):3435–3440. doi:10.1016/j.febslet.2014.07.036
Forsberg Z, Vaaje-Kolstad G, Westereng B, Bunaes AC, Stenstrom Y, MacKenzie A, Sorlie M, Horn SJ, Eijsink VGH (2011) Cleavage of cellulose by a CBM33 protein. Protein Sci 20(9):1479–1483. doi:10.1002/Pro.689
Westereng B, Ishida T, Vaaje-Kolstad G, Wu M, Eijsink VG, Igarashi K, Samejima M, Stahlberg J, Horn SJ, Sandgren M (2011) The putative endoglucanase PcGH61D from phanerochaete chrysosporium is a metal-dependent oxidative enzyme that cleaves cellulose. PLoS One 6(11):e27807. doi:10.1371/journal.pone.0027807
Phillips CM, Beeson WT, Cate JH, Marletta MA (2011) Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by neurospora crassa. ACS Chem Biol 6(12):1399–1406. doi:10.1021/cb200351y
Quinlan RJ, Sweeney MD, Lo Leggio L, Otten H, Poulsen JCN, Johansen KS, Krogh KBRM, Jorgensen CI, Tovborg M, Anthonsen A, Tryfona T, Walter CP, Dupree P, Xu F, Davies GJ, Walton PH (2011) Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci U S A 108(37):15079–15084. doi:10.1073/pnas.1105776108
Westereng B, Agger JW, Horn SJ, Vaaje-Kolstad G, Aachmann FL, Stenstrom YH, Eijsink VG (2013) Efficient separation of oxidized cello-oligosaccharides generated by cellulose degrading lytic polysaccharide monooxygenases. J Chromatogr A 1271(1):144–152. doi:10.1016/j.chroma.2012.11.048
Westereng B, Arntzen MØ, Aachmann FL, Várnai A, Eijsink VGH, Agger JW (2016) Simultaneous analysis of C1 and C4 oxidized oligosaccharides, the products of lytic polysaccharide monooxygenases acting on cellulose. J Chromatogr A 1445:46–54 http://dx.doi.org/10.1016/j.chroma.2016.03.064
Coenen GJ, Bakx EJ, Verhoef RP, Schols HA, Voragen AGJ (2007) Identification of the connecting linkage between homo- or xylogalacturonan and rhamnogalacturonan type I. Carbohydr Polym 70(2):224–235
Westereng B, Coenen GJ, Michaelsen TE, Voragen AGJ, Samuelsen AB, Schols HA, Knutsen SH (2009) Release and characterization of single side chains of white cabbage pectin and their complement-fixing activity. Mol Nutr Food Res 53(6):780–789. doi:10.1002/mnfr.200800199
Isaksen T, Westereng B, Aachmann FL, Agger JW, Kracher D, Kittl R, Ludwig R, Haltrich D, Eijsink VG, Horn SJ (2014) A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides. J Biol Chem 289(5):2632–2642. doi:10.1074/jbc.M113.530196
Agger JW, Isaksen T, Varnai A, Vidal-Melgosa S, Willats WG, Ludwig R, Horn SJ, Eijsink VG, Westereng B (2014) Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation. Proc Natl Acad Sci U S A 111(17):6287–6292. doi:10.1073/pnas.1323629111
Bennati-Granier C, Garajova S, Champion C, Grisel S, Haon M, Zhou S, Fanuel M, Ropartz D, Rogniaux H, Gimbert I, Record E, Berrin JG (2015) Substrate specificity and regioselectivity of fungal AA9 lytic polysaccharide monooxygenases secreted by podospora anserina. Biotechnol Biofuels 8:90. doi:10.1186/s13068-015-0274-3
Vu VV, Beeson WT, Span EA, Farquhar ER, Marletta MA (2014) A family of starch-active polysaccharide monooxygenases. Proc Natl Acad Sci U S A 111(38):13822–13827. doi:10.1073/pnas.1408090111
Lo Leggio L, Simmons TJ, Poulsen JC, Frandsen KE, Hemsworth GR, Stringer MA, von Freiesleben P, Tovborg M, Johansen KS, De Maria L, Harris PV, Soong CL, Dupree P, Tryfona T, Lenfant N, Henrissat B, Davies GJ, Walton PH (2015) Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase. Nat Commun 6:5961. doi:10.1038/ncomms6961
Frommhagen M, Sforza S, Westphal AH, Visser J, Hinz SWA, Koetsier MJ, van Berkel WJH, Gruppen H, Kabel MA (2015) Discovery of the combined oxidative cleavage of plant xylan and cellulose by a new fungal polysaccharide monooxygenase. Biotechnol Biofuels 8:12. doi:10.1186/s13068-015-0284-1
Kracher D, Scheiblbrandner S, Felice AKG, Breslmayr E, Preims M, Ludwicka K, Haltrich D, Eijsink VGH, Ludwig R (2016) Extracellular electron transfer systems fuel cellulose oxidative degradation. Science. doi:10.1126/science.aaf3165
Westereng B, Cannella D, Agger JW, Jorgensen H, Andersen ML, Eijsink VGH, Felby C (2015) Enzymatic cellulose oxidation is linked to lignin by long-range electron transfer. Sci Rep-Uk 5. doi: 10.1038/srep18561 ARTN 18561
Cannella D, Mollers KB, Frigaard NU, Jensen PE, Bjerrum MJ, Johansen KS, Felby C (2016) Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme. Nat Commun 7. doi:10.1038/ncomms11134
Eibinger M, Ganner T, Bubner P, Rosker S, Kracher D, Haltrich D, Ludwig R, Plank H, Nidetzky B (2014) Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency. J Biol Chem 289(52):35929–35938. doi:10.1074/jbc.M114.602227
Potthast A, Radosta S, Saake B, Lebioda S, Heinze T, Henniges U, Isogai A, Koschella A, Kosma P, Rosenau T, Schiehser S, Sixta H, Strlic M, Strobin G, Vorwerg W, Wetzel H (2015) Comparison testing of methods for gel permeation chromatography of cellulose: coming closer to a standard protocol. Cellul 22(3):1591–1613. doi:10.1007/s10570-015-0586-2
Beeson WT, Vu VV, Span EA, Phillips CM, Marletta MA (2015) Cellulose degradation by polysaccharide monooxygenases. Annu Rev Biochem 84:923–946. doi:10.1146/annurev-biochem-060614-034439
Hemsworth GR, Johnston EM, Davies GJ, Walton PH (2015) Lytic polysaccharide monooxygenases in biomass conversion. Trends Biotechnol 33(12):747–761. doi:10.1016/j.tibtech.2015.09.006
Hemsworth GR, Davies GJ, Walton PH (2013) Recent insights into copper-containing lytic polysaccharide mono-oxygenases. Curr Opin Struct Biol 23(5):660–668. doi:10.1016/j.sbi.2013.05.006
Cancilla MT, Wang AW, Voss LR, Lebrilla CB (1999) Fragmentation reactions in the mass spectrometry analysis of neutral oligosaccharides. Anal Chem 71(15):3206–3218. doi:10.1021/Ac9813484
Acknowledgments
This work was supported by the Norwegian Research Council through grants and 193817, 214138, 214613, 216162, 243663, and 244259.
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Westereng, B., Arntzen, M.Ø., Agger, J.W., Vaaje-Kolstad, G., Eijsink, V.G.H. (2017). Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry. In: Abbott, D., Lammerts van Bueren, A. (eds) Protein-Carbohydrate Interactions. Methods in Molecular Biology, vol 1588. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6899-2_7
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DOI: https://doi.org/10.1007/978-1-4939-6899-2_7
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