Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role
Christos Kosinas, Anastasia Zerva, Evangelos Topakas and Maria Dimarogona [1]
Molecular Tour
This study presents the crystal structure of a novel laccase-like multicopper oxidase from a thermophilic fungus (Thermothelomyces thermophila), named TtLMCO1. Multicopper oxidases are biocatalysts with various biotechnological applications, such as wastewater treatment, synthesis of novel bioactive compounds, lignin degradation as well as biosensors development. This study is focused on TtLMCO1, which is capable of oxidizing both ascorbic acid and phenolic compounds and is thus functionally categorized between ascorbate oxidases and fungal laccases. The crystal structure of TtLMCO1, determined using an AlphaFold 2.0 model in lack of experimentally determined structures of close homologues, revealed a three-domain laccase with two copper sites, lacking the C-terminal plug observed in other asco-laccases. The refined crystal structure of TtLMCO1 contains 576 residues arranged in three cupredoxin-like domains. Domain A includes residues 11 to 134, domain B includes residues 135-372 and domain C includes residues 373-602. A unique disulfide bond is formed between Cys28 and Cys236, stabilizing the relative orientation between domains A and B. Each domain (A,B,C) is colored differently, with cyan, dark red and green respectively. The disulfide bond between residues Cys28 and Cys236 is in stick representation. Copper ions and a dioxygen molecule located at the trinuclear cluster (TNC) site are shown as brown and red spheres respectively. Glycans are depicted as cyan sticks. A NAG
molecule bound with Asn37. A hydrogen bond is formed the main chain carbonyl oxygen of Ile16 and N2 atom of NAG (white dotted line). Two NAG molecules bound with Asn65. A hydrogen bond is formed between OD1 atom of Asp420 and N2 atom of NAG (white dotted line). A CH-pi interaction is formed between the phenyl group of Tyr595 and the D-glycopyranose ring of NAG.
Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Graphical representation of TtLMCO1 tunnels. T2 and T3 tunnels are shown as red and blue spheres, respectively. Copper ions are shown as brown spheres and side chains of residues forming the tunnels as shown as wheat sticks. Docking simulations with substrates that are oxidized by TtLMCO1 provide evidence that substrate specificity of these metallo-proteins is not exclusively related to their redox potential but also on the architecture of the binding site and the side chain flexibility of specific amino acids. Graphical representation of docking simulation results for TtLMCO1:
Also, our analysis points to a potential biological role of these biocatalysts, involving the production of pigments and other bioactive compounds in their natural hosts.
References
- ↑ Kosinas C, Zerva A, Topakas E, Dimarogona M. Structure-function studies of a novel laccase-like multicopper oxidase from Thermothelomyces thermophila provide insights into its biological role. Acta Crystallogr D Struct Biol. 2023 Jul 1;79(Pt 7):641-654. PMID:37326583 doi:10.1107/S2059798323004175
