Structural Biology: Lytic Polysaccharide Monooxygenases

Contact: Flora Meilleur (NCSU/ORNL) meilleurf@ornl.gov


The copper active site of NcLPM09D. A: Neutron
scattering length density maps revealing the
conformation and protonation state of 
His157. B. Hydrogen bond network conencting
the 2nd shell residues H157 and Q166 to Tyr168.
C: Electron density maps of the NcLPM09D
resting state revealing pre-binding of molecular
dioxygen in a distal pocket. D: Electron density
maps of ascorbate treated NcLPD09D 
revealing the activation of the dioxygen species.

Lytic polysaccharide monooxygenases (LPMOs) are copper containing enzymes produced by bacteria, fungi, insects and viruses to degrade polysaccharides, including cellulose and chitin. Since their discovery in 2010, LPMOs have generated immense interest and have been associated with i) cellulose degradation by fungi and insects, ii) viral pathogenicity (insect poxviruses) and iii) bacterial virulence (Listeria monocytogenes and Vibrio cholerae). However, precise details of the copper chemistry carried out by LPMOs to oxidize C—H bonds remains elusive. Elucidating how LPMOs function will have impacts on a broad range of applications that expand beyond biofuels to food security and drug design.

Using the model LPMOs NcLPMO9D and LsLPMO9A, we combine neutron crystallography and quantum mechanical modeling with advanced deuterium labeling and anerobic expression to address poorly understood aspects of the LPMO’s reaction mechanism.

Challenges we are working on

LPMOs bind and activate a dioxygen species at their Cu-containing active site to catalyze the reductive scission of the O—O bond, facilitating the insertion of one oxygen atom into the carbohydrate substrate molecule with formation of a water molecule. While described as monooxygenases, due to their binding of molecular dioxygen as co-substrate, recent findings demonstrate that LPMOs also have peroxidase activity and can preferentially utilize H2O2 over O2 depending on experimental conditions.

Neutron crystallography is the only structural technique that can unambiguously distinguish the oxygen containing species (e.g. O2, H2O2, —OOH, —O2) coordinated to or near the active site copper ion, which is highly susceptible to X-ray photoreduction.

We combine neutron structure determination with QM/MM calculation and deuterium labelling, to provide a complete, atomic-level, experimentally-informed, paradigm for the elucidation of the catalytic mechanism(s) of LPMOs and deliver the new level of understanding of these intriguing enzymes.

Building from our earlier work, we are specifically addressing the following questions: 1) What are the structural determinants of the H2O2 based mechanism? 2) How do the conserved second shell residues and other distant residues contribute to activity? 3) Does the O2 based mechanism follow a superoxyl, hydroperoxyl or oxyl catalytic pathway?