Electron supply

The enzymatic oxidation of polysaccharides by LPMOs depends on the external supply of electrons. Several sources of electrons have been shown to activate these enzymes since their discovery. LPMO activity was observed in the presence of small molecule reductants such as ascorbic acid, reduced glutathione (Vaaje-Kolstad et al., 2010) and gallic acid (Quinlan et al., 2011). A plethora of other reducing agents was found to be able to stimulate LPMOs in subsequent studies, amongst others cysteine, pyrogallol (Lo Leggio et al., 2015), resveratrol, catechin, caffeic acid and synaptic acid (Westereng et al., 2015).

Harris and colleagues observed that an AA9 (then known as GH61) from T. terrestris promoted degradation of lignocellulosic biomass, but not purified cellulose, by cellulases.

They did so prior to the discovery of the redox enzyme character of LPMOs (Harris et al., 2010). This observation was later confirmed and explained by other authors who showed that the respective LPMOs can be activated by lignin (Cannella et al., 2012, Dimarogona et al., 2012), a compound abundantly available in lignocellulosic biomass. The electron supply by lignin has been suggested to take place via long-range electron transfer where soluble low molecular weight lignin compounds shuttle electrons from high molecular weight lignin to the LPMO (Westereng et al., 2015).

Interestingly, other groups of redox-active enzymes are able to act as a reductant for LPMOs.

Cellobiose dehydrogenases (CDH; Figure 21) are flavocytochromes that can be found in

fungal secretomes (Henriksson et al., 2000, Phillips et al., 2011b, Kracher et al., 2016). CDH

contains two prosthetic groups, a flavin containing two electrons and a haem containing one

electron when the enzyme is fully reduced (Igarashi et al., 2002). The flavin domain, also

referred to as the dehydrogenase (DH) domain, belongs to the glucose-methanol-choline

(GMC) oxidoreductases and is classified as an AA3, whereas the haem b-binding cytochrome

(CYT) domain belongs to family AA8 as classified in the CAZy database (Levasseur et al.,

2013).

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Figure 21. Structure of MtCDH (PDB ID 4QI6) in the ‘closed’ state that allows IET between the cytochrome domain (magenta) and the dehydrogenase domain (blue). The CBM1 domain is shown in yellow and the flexible linker that did not crystallize, is shown as green dotted line. The figure was made with PyMol (DeLano and Lam, 2005).

The oxidation of cellobiose (and other substrates) to 1-5-δ-lactones is catalyzed by the DH

domain. The CYT domain acquires electrons via interdomain electron transfer (IET) from the

reduced DH domain to the haem (Tan et al., 2015). Tan et al. observed that CDH is present

in two conformations in solution, in the ‘closed’ and the ‘open’ state. The interaction between

the CYT domain and the DH domain in the ‘closed’ state is important for efficient IET

whereas the open state allows ET to an external electron acceptor like an LPMO (Tan et al.,

2015). Figure 21 shows the closed conformation of MtCDH. A reduced CYT domain is able

to reduce a wide range of substrates like metal ions, quinones or oxygen (Phillips et al.,

2011a). In a knockout study, Phillips et al. (2011a) showed the importance of CDH for

cellulose degradation by N. crassa. The culture supernatant of the knockout strain was

significantly less efficient in the degradation of Avicel, but reached wildtype activity when

external CDH was added. The same study also reported that CDH was able to serve as an

electron donor for three different LPMOs from N. crassa (Phillips et al., 2011a) and suggested

that this was likely to be the biologically relevant role of CDH. Thus, AA9-type LPMOs are

not only activated by small molecule reductants, but also by CDHs (Phillips et al., 2011a,

Langston et al., 2011, Beeson et al., 2012). Figure 22 illustrates the activation of LPMOs by

CDH. The interaction between CDH and fungal LPMOs has been suggested to occur via a

conserved surface patches co-evolved on both enzymes (Li et al., 2012). The same authors

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suggested that ET takes place through long distance electron transfer via a conserved hydrogen bond network or conserved aromatic residues (Li et al., 2012). In contrast, the authors of a recent NMR study showed that interaction between a CDH and an LPMO occurs directly at the copper active site (Courtade et al., 2016), thus indicating the absence of a conserved site for ET on the LPMO. So far, CDHs and related proteins have only been found in fungi. Bacteria do not seem to encode a protein analogous to CDH. However, a large multimodular protein with predicted cytochrome domains (i.e. redox activities) identified in Cellvibrio japonicus was indeed able to activate an LPMO (Gardner et al., 2014). It is thus possible that bacteria produce proteins that are able to provide electrons to secreted LPMOs.

Fungal CDHs are not the only redox active proteins that contribute to LPMO activation.

Kracher et al. (2016) recently showed that a variety of plant-derived or fungal diphenols can efficiently reduce an AA9 from Neurospora crassa, but that they are irreversibly depleted in the process. However, regeneration of these reductants could be achieved by addition of GMC oxidoreductases, implying that the phenolic compounds can act as a redox mediators between GMC oxidoreductases and LPMOs. In another very recent study, Garajova et al. (2016) observed that flavoenzymes of family AA3 are also able to directly interact with AA9s, extending the array of LPMO stimulating protein based reductants.

Other researchers have used quite different approaches to reduce LPMOs. By using the energy of light Cannella and co-workers were able to activate an AA9-type LPMO in the presence of a pigment, either thylakoids or chlorophyllin, and an electron source such as ascorbate and lignin. By using this system the authors claim an up to two orders of magnitude increase in catalytic activity compared to previously reported values (Cannella et al., 2016). In another study Bissaro et al. (2016) report that vanadium-doped TiO

can be used to activate AA10s.

The photocatalyst catalyzes the light-driven oxidation of water, thereby providing electrons

to the LPMO. Notably, these two light-driven scenarios are quite different. The first yields

much higher LPMO activity, but relies on externally added reducing equivalents. In contrast,

the second relies on light and catalyst only, but gives low activity.

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Figure 22. Lytic polysaccharide oxidation by the CDH-LPMO system. An oxidized CDH (square)

acquires electrons from a substrate (here: lactose). The reduced CDH then transfers the electrons to an

oxidized Cu(II)-LPMO (triangle) and thereby, gets re-oxidized. The reduced LPMO activates

dioxygen and then oxidatively cleaves a substrate thus, it gets re-oxidized. In the absence of an intact

LPMO or an LPMO substrate, CDH or the LPMO respectively transfer electrons to dissolved O

which

results in the formation of hydrogen peroxide. Enzymes are colored blue in their oxidized form and

pink in their reduced form. The figure was taken from [paper II in this thesis (Loose et al., submitted)].