Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase

Abstract

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O 2 − . Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action.

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

“…The same applies to An 3046, although this was not noted by the authors (16). The L3 loop has been proposed to be a structural determinant of xyloglucan activity (14), and a recent nuclear magnetic resonance (NMR) study of enzyme-substrate interactions in Nc LPMO9C showed that the L3 loop indeed interacts with xyloglucan (55). The present data and data for An 3046 show that xyloglucan cleavage can also be achieved by LPMOs lacking the L3 loop.…”

Oxygen Activation by Cu LPMOs in Recalcitrant Carbohydrate Polysaccharide Conversion to Monomer Sugars

“…Much of what is known about the LPMO–ligand interaction stems from studies on LPMOs that are active on soluble substrates, though there is a study of insoluble β -chitin binding to CBP21/ Sm AA10A using NMR and an indirect method utilizing 2 H/ 1 H exchange. 180,196,249 There are also site-directed mutagenesis studies in which the effect of selected amino acid residues on the catalytic activity and/or substrate binding have been probed. 95,110,183 In an early publication on Sm LPMO10A (CBP21), Vaaje-Kolstad and co-workers showed that the single aromatic residue on the proposed binding surface is important for chitin binding.…”

“…180 The electronic nature of the interaction may explain the large effect on 15 N chemical shift reported for His-1 of Nc LPMO9C. 249 Beneath the –1 subsite in LsAA9A structure complexes is a conserved glutamine residue interacting with a chloride ion that is localized near the substrate. This glutamine is conserved not only in AA9 but also in AA13 LPMOs.…”

“…Two separate studies have since suggested a direct interaction between the cytochrome domain and the copper atom in the active site. 172,249 In the first study, Tan et al determined the structures of full-length CDH and the LPMO9F from N. crassa as well as the structure of the CDH from M. thermophilum. 172 By measuring the kinetics of the electron transfer from the Nc CDH cytochrome domain to Nc LPMO9F and comparing with stopped-flow experiments where the flavin domain was included, they concluded that the interaction is between the Nc CDH cytochrome domain and Nc LPMO9F.…”

Lignocellulases: a review of emerging and developing enzymes, systems, and practices

“…Nevertheless, their functional distinctions and associated mechanisms are yet to be fully elucidated to help exploit their maximum benefits. Accordingly, studies focusing on the structure (Harris et al 2010;Aachmann et al 2012;Hemsworth et al 2013b;Borisova et al 2015;Frandsen et al 2016) and interactions (Isaksen et al 2014;Eibinger et al 2014;Courtade et al 2016;Kracher et al 2016) of LPMOs are surfacing.…”