Backbone and side-chain 1H, 13C, and 15N chemical shift assignments for the apo-form of the lytic polysaccharide monooxygenase NcLPMO9C

Abstract

The apo-form of the 23.3 kDa catalytic domain of the AA9 family lytic polysaccharide monooxygenase NcLPMO9C from Neurospora crassa has been isotopically labeled and recombinantly expressed in Pichia pastoris. In this paper, we report the 1 H, 13 C, and 15 N chemical shift assignments of this LPMO.

Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates

“…Recent ITC, NMR and docking studies of an AA9 LPMO from Neurospora crassa in contact with oligosaccharides revealed that more extended substrates had significantly higher binding affinities. This is in accord with a multi-point interaction of the substrate with the LPMO surface where the surface loops in some LPMOs remote from the active site enhance binding affinity 21 . The study also showed that a single cellohexaose (Cell 6 ) chain likely spans the copper active site from the −3 to +3 or −2 to +4 subsites (subdivisions of the binding cleft numbered relative to the site of cleavage 32 ), in which the L3 loop (important for interactions with the +3/+4 subsites) and the LC loop (important for binding to approximately −4 subsite) lie at somewhat extended distances from the copper active site.…”

“…We studied substrate binding on both Ls AA9A and Cv AA9A using EPR spectroscopy (Table 3 ) to investigate the electronic state of the active site copper upon binding. As has been shown by Frandsen et al 33 and Courtade et al 21 , the binding affinity of oligosaccharide substrates is significantly affected by the presence of the exogenous ligand on the copper ion. Accordingly, EPR experiments were carried out in both the absence and presence of 200 mM chloride (1.0 M chloride for Xyl 6 studies).…”

“…Data were collected on crystals soaked in GM ( Ls AA9A:GM; PDB 5NKW) or XG stock solutions (in 3.8 M NaCl, 0.1 M citric acid pH 5.5). Crystals were also soaked in the presence of 0.3 M XXXG, and up to 1.2 M of XG oligosaccharide purchased from Megazyme (consisting primarily of XXXGXXXG, see Courtade et al 21 ). No complex structures were obtained from any of the crystals soaked with XG substrates, either because no binding was observed or only cellooligosaccharides were bound (presumably because acid hydrolysis caused debranching).…”

A fast and sensitive activity assay for lytic polysaccharide monooxygenase

“…Nevertheless, if suitable reaction conditions, sampling time points and correct calibration data for the detected species are used, HPLC-based methods provide the most accurate results of physiological LPMO activity. NMR has also been used to follow the action of LPMO on polymeric substrates [ 4 , 6 , 11 , 12 ], but its strength lies in the identification of reaction products and not in the measurement of enzymatic activity.…”

Resonance assignments for the apo-form of the cellulose-active lytic polysaccharide monooxygenase TaLPMO9A

“…Because LPMO action increases the susceptibility of recalcitrant substrates such as cellulose and chitin to the action of classical glycoside hydrolases (GHs), LPMOs have become an important ingredient in commercial enzyme cocktails for industrial biomass conversion (Hu et al 2014;Müller et al 2015). To create additional insight into LPMO structure and dynamics, and to study substrate binding, several NMR investigations have been conducted (Aachmann et al 2011(Aachmann et al , 2012Courtade et al 2015Courtade et al , 2016aCourtade et al , b, 2017.…”