Format

Send to

Choose Destination
J Biol Chem. 2018 Jan 26;293(4):1397-1412. doi: 10.1074/jbc.M117.817130. Epub 2017 Dec 8.

Structural determinants of bacterial lytic polysaccharide monooxygenase functionality.

Author information

1
From the Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway, zarah.forsberg@nmbu.no.
2
From the Faculty of Chemistry, Biotechnology, and Food Science, Norwegian University of Life Sciences (NMBU), 1432 Ås, Norway.
3
INRA, UMR792, Ingénierie des Systèmes Biologiques et des Procédés, F-31400 Toulouse, France, and.
4
the Department of Medical Biochemistry, Institute for Clinical Medicine, University of Oslo, 0318 Oslo, Norway, and.
5
the Department of Microbiology, Clinic for Laboratory Medicine, Oslo University Hospital, Rikshospitalet, P. O. Box 4950, Nydalen, N-0424 Oslo, Norway.

Abstract

Bacterial lytic polysaccharide monooxygenases (LPMO10s) use redox chemistry to cleave glycosidic bonds in the two foremost recalcitrant polysaccharides found in nature, namely cellulose and chitin. Analysis of correlated mutations revealed that the substrate-binding and copper-containing surface of LPMO10s composes a network of co-evolved residues and interactions, whose roles in LPMO functionality are unclear. Here, we mutated a subset of these correlated residues in a newly characterized C1/C4-oxidizing LPMO10 from Micromonospora aurantiaca (MaLPMO10B) to the corresponding residues in strictly C1-oxidizing LPMO10s. We found that surface properties near the catalytic copper, i.e. side chains likely to be involved in substrate positioning, are major determinants of the C1:C4 ratio. Several MaLPMO10B mutants almost completely lost C4-oxidizing activity while maintaining C1-oxidizing activity. These mutants also lost chitin-oxidizing activity, which is typically observed for C1/C4-oxidizing, but not for C1-oxidizing, cellulose-active LPMO10s. Selective loss in C1-oxidizing activity was not observed. Additional mutational experiments disclosed that neither truncation of the MaLPMO10B family 2 carbohydrate-binding module nor mutations altering access to the solvent-exposed axial copper coordination site significantly change the C1:C4 ratio. Importantly, several of the mutations that altered interactions with the substrate exhibited reduced stability. This effect could be explained by productive substrate binding that protects LPMOs from oxidative self-inactivation. We discuss these stability issues in view of recent findings on LPMO catalysis, such as the involvement of H2O2 Our results show that residues on the substrate-binding surface of LPMOs have co-evolved to optimize several of the interconnected properties: substrate binding and specificity, oxidative regioselectivity, catalytic efficiency, and stability.

KEYWORDS:

LPMO; cellulose; chitin; hydrogen peroxide; protein evolution; regioselectivity; site-directed mutagenesis; substrate specificity

PMID:
29222333
PMCID:
PMC5787815
DOI:
10.1074/jbc.M117.817130
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central Icon for Norwegian BIBSYS system
Loading ...
Support Center