Spectroscopic and Structural Studies on Termite LPMOs

Abstract

Termites are social insects that play a fundamental role in carbon cycling in tropical forests and savannas. They display labor division among their castes and are highly efficient in lignocellulose degradation. Our group has described that oxidative enzymes could be involved in plant biomass digestion in lower termite guts along with glycoside hydrolases. Additionally, unpublished data from our group disclosed in the Coptotermes gestroi genome two genes containing the LPMO_10 Pfam domain (PF03067), which encode for oxidative copper-dependent enzymes known as lytic polysaccharide monooxygenases or LPMOs. These metalloenzymes can cleave cellulose, hemicellulose, starch, and chitin polymers by oxidative mechanism instead of the classic hydrolytic mechanism observed for cellulases and hemicellulases. After the search for clusters of orthologous LPMOs in the EggNOG database, using the C. gestroi LPMO sequences as model sequences, it was retrieved more than 80 putative metazoan/animal LPMOs, especially within the Arthropoda phylum. The phylogenetic analysis revealed that the termite LPMO genes, as well as their metazoan orthologous, did not cluster with characterized LPMOs from families AA9, AA10, AA11, and AA13, suggesting novel LPMO family members. Thus, the aim of this study is to investigate and to compare the structural and spectroscopic features from two phylogenetic distinct C. gestroi LMPOs (CgLPMO-1 and CgLPMO-2), focusing on the histidine-copper brace in its activity site and the interactions with the substrates. Using a combination of different techniques such as displacement isothermal titration calorimetry (ITC), differential scanning fluorimetry (DSF), electron paramagnetic resonance (EPR) and protein-ligand crystallography, the focus will be to compare the interactions between enzyme-copper ion, as well as enzyme-substrate. Consequently, these studies will contribute to a better understanding of the molecular basis of polysaccharide cleavage by novel LPMOs belonging to new AA families.