posted on 2024-05-15, 14:06authored byKelsi
R. Hall, Maja Mollatt, Zarah Forsberg, Ole Golten, Lorenz Schwaiger, Roland Ludwig, Iván Ayuso-Fernández, Vincent G. H. Eijsink, Morten Sørlie
Lytic polysaccharide
monooxygenases (LPMOs) catalyze the oxidative
cleavage of glycosidic bonds in recalcitrant polysaccharides, such
as cellulose and chitin, using a single copper cofactor bound in a
conserved histidine brace with a more variable second coordination
sphere. Cellulose-active LPMOs in the fungal AA9 family and in a subset
of bacterial AA10 enzymes contain a His-Gln-Tyr second sphere motif,
whereas other cellulose-active AA10s have an Arg–Glu–Phe
motif. To shine a light on the impact of this variation, we generated
single, double, and triple mutations changing the His216–Gln219–Tyr221 motif in cellulose-
and chitin-oxidizing MaAA10B toward Arg–Glu–Phe.
These mutations generally reduced enzyme performance due to rapid
inactivation under turnover conditions, showing that catalytic fine-tuning
of the histidine brace is complex and that the roles of these second
sphere residues are strongly interconnected. Studies of copper reactivity
showed remarkable effects, such as an increase in oxidase activity
following the Q219E mutation and a strong dependence of this effect
on the presence of Tyr at position 221. In reductant-driven reactions,
differences in oxidase activity, which lead to different levels of
in situ generated H2O2, correlated with differences
in polysaccharide-degrading ability. The single Q219E mutant displayed
a marked increase in activity on chitin in both reductant-driven reactions
and reactions fueled by exogenously added H2O2. Thus, it seems that the evolution of substrate specificity in LPMOs
involves both the extended substrate-binding surface and the second
coordination sphere.