期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2020
卷号：117
期号：32
页码：19178-19189
DOI：10.1073/pnas.2004277117
出版社：The National Academy of Sciences of the United States of America
摘要：Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO–substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo - Bl LPMO10A from Bacillus licheniformis , along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15 N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63 Cu(II)-bound 15 N- Bl LPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal–ligand covalency and an attendant increase in the d(x 2 −y 2 ) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu–O 2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O 2 activation in chitin-active AA10 LPMOs.
