posted on 2018-03-02, 00:00authored byBastien Bissaro, Ingvild Isaksen, Gustav Vaaje-Kolstad, Vincent G. H. Eijsink, Åsmund K. Røhr
Lytic
polysaccharide monooxygenases (LPMOs) are major players in
biomass conversion, both in Nature and in the biorefining industry.
How the monocopper LPMO active site is positioned relative to the
crystalline substrate surface to catalyze powerful, but potentially
self-destructive, oxidative chemistry is one of the major questions
in the field. We have adopted a multidisciplinary approach, combining
biochemical, spectroscopic, and molecular modeling methods to study
chitin binding by the well-studied LPMO from Serratia marcescens
SmAA10A (or CBP21). The orientation of the enzyme on a single-chain
substrate was determined by analyzing enzyme cutting patterns. Building
on this analysis, molecular dynamics (MD) simulations were performed
to study interactions between the LPMO and three different surface
topologies of crystalline chitin. The resulting atomistic models showed
that most enzyme–substrate interactions involve the polysaccharide
chain that is to be cleaved. The models also revealed a constrained
active site geometry as well as a tunnel connecting the bulk solvent
to the copper site, through which only small molecules such as H2O, O2, and H2O2 can diffuse.
Furthermore, MD simulations, quantum mechanics/molecular mechanics
calculations, and electron paramagnetic resonance spectroscopy demonstrate
that rearrangement of Cu-coordinating water molecules is necessary
when binding the substrate and also provide a rationale for the experimentally
observed C1 oxidative regiospecificity of SmAA10A.
This study provides a first, experimentally supported, atomistic view
of the interactions between an LPMO and crystalline chitin. The confinement
of the catalytic center is likely crucially important for controlling
the oxidative chemistry performed by LPMOs and will help guide future
mechanistic studies.