Journal:Acta Cryst D:S2059798323004175

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<b>Molecular Tour</b><br>
<b>Molecular Tour</b><br>
This study presents the crystal structure of a novel laccase-like multicopper oxidase from a thermophilic fungus (''Thermothelomyces thermophila''), named ''Tt''LMCO1. 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 ''Tt''LMCO1, which is capable of oxidizing both ascorbic acid and phenolic compounds and is thus functionally categorized between ascorbate oxidases and fungal laccases. <scene name='96/965740/Overall/2'>The crystal structure of TtLMCO1</scene>, 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. <scene name='96/965740/Domain_a/1'>Domain A</scene> includes residues 11 to 134, <scene name='96/965740/Domain_b/1'>domain B</scene> includes residues 135-372 and <scene name='96/965740/Domain_c/1'>domain C</scene> includes residues 373-602. <scene name='96/965740/Disulfide_bond/1'>A unique disulfide bond is formed between Cys28 and Cys236</scene>, 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 <scene name='96/965740/Trinuclear_cluster/1'>the trinuclear cluster (TNC) site</scene> are shown as brown and red spheres respectively. Glycans are depicted as cyan sticks. <scene name='96/965740/Nag_molecule_n37/1'>A NAG
This study presents the crystal structure of a novel laccase-like multicopper oxidase from a thermophilic fungus (''Thermothelomyces thermophila''), named ''Tt''LMCO1. 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 ''Tt''LMCO1, which is capable of oxidizing both ascorbic acid and phenolic compounds and is thus functionally categorized between ascorbate oxidases and fungal laccases. <scene name='96/965740/Overall/2'>The crystal structure of TtLMCO1</scene>, 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. <scene name='96/965740/Domain_a/1'>Domain A</scene> includes residues 11 to 134, <scene name='96/965740/Domain_b/1'>domain B</scene> includes residues 135-372 and <scene name='96/965740/Domain_c/1'>domain C</scene> includes residues 373-602. <scene name='96/965740/Disulfide_bond/1'>A unique disulfide bond is formed between Cys28 and Cys236</scene>, 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 <scene name='96/965740/Trinuclear_cluster/1'>the trinuclear cluster (TNC) site</scene> are shown as brown and red spheres respectively. Glycans are depicted as cyan sticks. <scene name='96/965740/Nag_molecule_n37/1'>A NAG
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molecule bound with Asn37</scene>. A hydrogen bond is formed the main chain carbonyl oxygen of Ile16 and N2 atom of NAG (white dotted line). <scene name='96/965740/Nag_molecules_n65/2'>Two NAG molecules bound with Asn65</scene>. A hydrogen bond is formed between OD1 atom of <scene name='96/965740/Nag_molecules_n65/3'>Asp420 and N2 atom NAG</scene> (white dotted line). A CH-pi interaction is formed between the <scene name='96/965740/Nag_molecules_n65/5'>phenyl group of Tyr595 and the D-glycopyranose ring of NAG</scene>.
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molecule bound with Asn37</scene>. A hydrogen bond is formed the main chain carbonyl oxygen of Ile16 and N2 atom of NAG (white dotted line). <scene name='96/965740/Nag_molecules_n65/2'>Two NAG molecules bound with Asn65</scene>. A hydrogen bond is formed between OD1 atom of <scene name='96/965740/Nag_molecules_n65/6'>Asp420 and N2 atom of NAG</scene> (white dotted line). A CH-pi interaction is formed between the <scene name='96/965740/Nag_molecules_n65/5'>phenyl group of Tyr595 and the D-glycopyranose ring of NAG</scene>.
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Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. Docking simulations with substrates that are oxidized by ''Tt''LMCO1 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. 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.
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Analysis of solvent tunnels highlighted the amino acids that are crucial for proton transfer into the trinuclear copper site. <scene name='96/965740/Ttlmco1_tunnels/10'>Graphical representation of TtLMCO1 tunnels</scene>. 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 ''Tt''LMCO1 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:
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*<scene name='96/965740/Binding_pocket/6'>Ball-and-stick representation of the substrate
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binding pocket of TtLMCO1 with L-ascorbic acid</scene>.
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**<jmol>
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<jmolLink>
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<script> select (atomno=8866),(atomno=5677),(atomno=8862),(atomno=5678),(atomno=5851),(atomno=8279),(atomno=8863),(atomno=6993),(atomno=6994),(atomno=4642),(atomno=4645),(atomno=8864),(atomno=2475); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrogen bonds.</text>
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</jmolLink>
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</jmol>
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**<jmol>
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<jmolLink>
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<script> select (atomno=8861),(atomno=9003),(atomno=9000),(atomno=2468); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrophobic interactions.</text>
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</jmolLink>
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</jmol>
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*<scene name='96/965740/Binding_pocket/7'>Ball-and-stick representation of the substrate binding pocket of TtLMCO1 with 2,6-DMP</scene>.
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**<jmol>
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<jmolLink>
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<script> select (atomno=8872),(atomno=5678),(atomno=5851),(atomno=8279),(atomno=8856),(atomno=8860); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrogen bonds.</text>
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</jmolLink>
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</jmol>
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**<jmol>
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<jmolLink>
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<script> select (atomno=8873),(atomno=9003),(atomno=9001); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrophobic interactions.</text>
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</jmolLink>
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</jmol>
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**<jmol>
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<jmolLink>
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<script> select (atomno=9008),(atomno=9007); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Pi-pi interactions.</text>
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</jmolLink>
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</jmol>
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*<scene name='96/965740/Binding_pocket/10'>Ball-and-stick representation of the substrate binding pocket of TtLMCO1 with ferulic acid.</scene>
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**<jmol>
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<jmolLink>
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<script> select (atomno=8877),(atomno=8879),(atomno=5678),(atomno=5851),(atomno=8279); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrogen bonds.</text>
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</jmolLink>
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</jmol>
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**<jmol>
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<jmolLink>
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<script> select (atomno=8857),(atomno=5818),(atomno=9002); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Hydrophobic interactions.</text>
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</jmolLink>
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</jmol>
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**<jmol>
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<jmolLink>
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<script> select (atomno=9009),(atomno=9007); selectionHalos ON;
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delay 0.5; selectionHalos OFF;</script>
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<text>Pi-pi interactions.</text>
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</jmolLink>
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</jmol>
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*<scene name='96/965740/Binding_pocket/11'>Spacefill representation of the substrate binding pocket of TtLMCO1 with L-ascorbic acid</scene>. {{Template:ColorKey_Hydrophobic}}, {{Template:ColorKey_Polar}}
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*<scene name='96/965740/Binding_pocket/12'>Spacefill representation of the substrate binding pocket of TtLMCO1 with 2,6-DMP</scene>.
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*<scene name='96/965740/Binding_pocket/13'>Spacefill representation of the substrate binding pocket of TtLMCO1 with ferulic acid</scene>.
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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.
<b>References</b><br>
<b>References</b><br>

Current revision

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Proteopedia Page Contributors and Editors (what is this?)

Alexander Berchansky, Jaime Prilusky

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