Activity, stability and 3-D structure of the Cu(ii) form of a chitin-active lytic polysaccharide monooxygenase from Bacillus amyloliquefaciens

Abstract

The enzymatic deconstruction of recalcitrant polysaccharide biomass is central to the conversion of these substrates for societal benefit, such as in biofuels. Traditional models for enzyme-catalysed polysaccharide degradation involved the synergistic action of endo-, exo-and processive glycoside hydrolases working in concert to hydrolyse the substrate. More recently this model has been succeeded by one featuring a newly discovered class of mononuclear copper enzymes: lytic polysaccharide monooxygenases (LPMOs; classified as Auxiliary Activity (AA) enzymes in the CAZy classification). In 2013, the structure of an LPMO from Bacillus amyloliquefaciens, BaAA10, was solved with the Cu centre photoreduced to Cu(I) in the X-ray beam. Here we present the catalytic activity of BaAA10. We show that it is a chitin-active LPMO, active on both α and β chitin, with the Cu(II) binding with low nM KD, and the substrate greatly increasing the thermal stability of the enzyme. A spiral data collection strategy has been used to facilitate access to the previously unobservable Cu(II) state of the active centre, revealing a coordination geometry around the copper which is distorted from axial symmetry, consistent with the previous findings from EPR spectroscopy.

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

“…Eleven of these are of bacterial origin and are functional in the oxidation of biomass. 120,121,123,124,183,196–201 There are also three proteins of viral origin classified as AA10. 80 These are all from insect poxviruses, in which the LPMO domain plays a role as part of the spindle protein fusolin, essential for the virus to infect its target insect and has been suggested to cause disruption of the chitin-rich peritrophic matrix in the insect gut (Figure 13D).…”

Production and spectroscopic characterization of lytic polysaccharide monooxygenases

“…The most common approach has been to secrete the resultant protein to the periplasm (Courtade et al, 2017;Forsberg et al, 2014a;Hemsworth et al, 2013b;Vaaje-Kolstad et al, 2005a. It is also possible, however, to express these enzymes in the cytoplasm using specialised E. coli strains (Forsberg et al, 2011(Forsberg et al, , 2014bGregory et al, 2016). These have largely been our methods of choice for LPMO production and though fungal enzymes are rarely produced using these strategies, we were able to express and purify an AA11 from Aspergillus oryzae using a periplasmic secretion system in E. coli (Hemsworth et al, 2014).…”

“…(Hemsworth et al, 2013b). c, SDS-PAGE gel showing stages from the purification of BaAA10 using a SUMOtagged construct (Gregory et al, 2016). If an apo-enzyme is not desired, but rather a copper loaded sample is to be used for study, prior to the final gel filtration step, CuCl2 can be added in a slight excess (typically 1.5x) in order to load the protein with copper prior to gel filtration.…”

“…Given that LPMOs are typically extracellular enzymes that contain disulfide bonds, the periplasmic expression protocol described above often represents the first port of call for protein production (Courtade et al, 2017;Forsberg et al, 2014a;Hemsworth et al, 2013b;Vaaje-Kolstad et al, 2005a. This is not the only option however, and there are reports of LPMOs being expressed intracellularly using cleavable tags at the N-terminus to aid purification (Forsberg et al, 2011(Forsberg et al, , 2014bGregory et al, 2016). In order to achieve this, the protease to be used to remove that tag must be carefully selected in order to leave the native N-terminal histidine available for copper binding.…”

“…Factor Xa (Forsberg et al, 2014b) and enterokinase are two such proteases. We prefer the use of a SUMO-tag (Champion-pET-SUMO, Invitrogen) which can be removed using SUMO-protease (Invitrogen) (Gregory et al, 2016). This method has the advantage that the SUMO-tag can act as a solubility enhancing tag in addition to providing an N-terminal histidine tag to aid in the purification of the protein.…”