Assessing the role of E451, D452, and H139 in the catalytic function of a Thermus thermophilus bacterial laccase
Por:
Miranda-Blancas R., Cardona-Echavarría M.C., Saab-Rincón G., Millán-Pacheco C., Landa A., Rudiño-Piñera E.
Publicada:
1 ene 2026
Ahead of Print:
1 ene 2026
Resumen:
Laccases are oxidoreductase enzymes that require four electrons and four protons for each catalytic cycle to trigger the conversion of one oxygen molecule into two water molecules. However, for bacterial laccases, including the laccase from Thermus thermophilus HB27 ( Tth -MCO), the complete proton transfer pathway from the solvent-exposed surface of the protein to the trinuclear center remains unclear. This paper describes new crystallographic structures of a partially copper-occupied Tth -MCO, subjected to a cumulative X-ray radiation strategy to uncover the effect of the X-ray driven chemical reduction on the residues related to the enzyme's proton relay. Additionally, we constructed and analyzed single and double mutants of residues H139 and E451 to assess their effect on the kinetic parameters of Tth -MCO. The proper folding of the mutants was evaluated using circular dichroism, while the structural stability of the mutants was analyzed using molecular dynamics calculations based on AlphaFold 2 models. Such analysis of the structural and kinetic data obtained provides deeper insights into the amino acids involved in the proton relay mechanism of Tth -MCO. Considering the conservation of amino acids involved in several bacterial laccase sequences, the proposed multi-step proton relay mechanism may also exist in other bacterial laccases. © 2026 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM).
Filiaciones:
Departamento de Microbiología y Parasitología, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad Universitaria, Ciudad de México, C. P. 04510, Mexico
Laboratorio de Biología Estructural, Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Morelos, Cuernavaca, 62210, Mexico
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, Morelos, Cuernavaca, 62210, Mexico
Facultad de Farmacia, Universidad Autónoma del Estado de Morelos., Morelos, Cuernavaca, 62209, Mexico
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