Analytical Tools for Characterizing Cellulose-Active Lytic Polysaccharide Monooxygenases (LPMOs)
Lytic polysaccharide monooxygenases are copper-dependent enzymes that perform oxidative cleavage of glycosidic bonds in cellulose and various other polysaccharides. LPMOs acting on cellulose use a reactive oxygen species to abstract a hydrogen from the C1 or C4, followed by hydroxylation of the resulting substrate radical. The resulting hydroxylated species is unstable, resulting in glycoside bond scission and formation of an oxidized new chain end. These oxidized chain ends are spontaneously hydrated at neutral pH, leading to formation of an aldonic acid or a gemdiol, respectively. LPMO activity may be characterized using a variety of analytic tools, the most common of which are high-performance anion exchange chromatography system with pulsed amperometric detection (HPAEC-PAD) and MALDI-TOF mass spectrometry (MALDI-MS). NMR may be used to increase the certainty of product identifications, in particular the site of oxidation. Kinetic studies of LPMOs have several pitfalls and to avoid these, it is important to secure copper saturation, avoid the presence of free transition metals in solution, and control the amount of reductant (i.e., electron supply to the LPMO). Further insight into LPMO properties may be obtained by determining the redox potential and by determining the affinity for copper. In some cases, substrate affinity can be assessed using isothermal titration calorimetry. These methods are described in this chapter.
Key wordsLytic polysaccharide monooxygenase High-performance anion-exchange chromatography MALDI-TOF mass spectrometry Copper Isothermal titration calorimetry
This work was supported by the Norwegian Research Council through grants 214613, 216162, 214138, 226244, 221576, 226247, and 244259.
- 4.Beeson WT, Vu VV, Span EA et al (2015) Cellulose degradation by polysaccharide monooxygenases. Annu Rev Biochem 84:923–946. https://doi.org/10.1146/annurev-biochem-060614-034439CrossRefPubMedGoogle Scholar
- 6.Bissaro B, Rohr AK, Muller G et al (2017) Oxidative cleavage of polysaccharides by monocopper enzymes depends on H2O2. Nat Chem Biol Adv 13(10):1123–1128. https://doi.org/10.1038/nchembio.2470. http://www.nature.com/nchembio/journal/vaop/ncurrent/abs/nchembio.2470.html#supplementary-informationCrossRefGoogle Scholar
- 26.Aachmann FL, Sorlie M, Skjak-Braek G et al (2012) NMR structure of a lytic polysaccharide monooxygenase provides insight into copper binding, protein dynamics, and substrate interactions. Proc Natl Acad Sci USA 109:18779–18784. https://doi.org/10.1073/pnas.1208822109CrossRefPubMedPubMedCentralGoogle Scholar
- 31.Vuong TV, Liu B, Sandgren M, Master ER (2017) Microplate-based detection of lytic polysaccharide monooxygenase activity by fluorescence-labeling of insoluble oxidized products. Biomacromolecules. https://doi.org/10.1021/acs.biomac.6b01790
- 44.Kojima Y, Varnai A, Ishida T et al (2016) Characterization of an LPMO from the brown-rot fungus Gloeophyllum trabeum with broad xyloglucan specificity, and its action on cellulose-xyloglucan complexes. Appl Environ Microbiol 82:6557–6572. https://doi.org/10.1128/AEM.01768-16CrossRefPubMedPubMedCentralGoogle Scholar