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Functional Characterization and Structural Analysis of NADH Oxidase Mutants from Thermus thermophilus HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability

1
Department of Biocatalysis, Institute of Catalysis and Petrochemistry (ICP) CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain
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Institute of Theoretical Chemistry, Faculty of Chemistry, University of Vienna, Währinger Str. 17 A-1090 Vienna, Austria
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Heterogeneous Biocatalysis Laboratory. Instituto de Síntesis Química y Catálisis Homogénea (ISQCH-CSIC), University of Zaragoza, 50009 Zaragoza, Spain
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ARAID, Aragon I+D Foundation, Av. de Ranillas 1-D, planta 2a, oficina B, 50018 Zaragoza, Spain
5
Department of Molecular Biology, Universidad Autónoma de Madrid, Center for Molecular Biology Severo-Ochoa (UAM-CSIC), Nicolás Cabrera 1, 28049 Madrid, Spain
*
Authors to whom correspondence should be addressed.
Microorganisms 2019, 7(11), 515; https://doi.org/10.3390/microorganisms7110515
Received: 15 October 2019 / Revised: 25 October 2019 / Accepted: 28 October 2019 / Published: 31 October 2019
(This article belongs to the Special Issue Extremophiles and Extremozymes in Academia and Industries)
The Thermus thermophilus strain HB27 NADH-oxidase (Tt27-NOX) catalyzes the oxidation of nicotinamide adenine dinucleotide (NAD(P)H) by reducing molecular oxygen to hydrogen peroxide in a two-electron transfer mechanism. Surprisingly, Tt27-NOX showed significant differences in catalytic properties compared to its counterpart from the strain HB8 (Tt8-NOX), despite a high degree of sequence homology between both variants. The sequence comparison between both enzymes revealed only three divergent amino acid residues at positions 166, 174, and 194. Motivated with these findings, in this work we performed mutagenesis experiments in the former three positions to study the specific role of these residues in the catalytic properties and thermostability of Tt27-NOX. We subjected five mutants, along with the wild-type enzyme, to biochemical characterization and thermal stability studies. As a result, we identified two more active and more thermostable variants than any Tt8-NOX variant reported in the literature. The most active and thermostable variant K166/H174/Y194 retained 90% of its initial activity after 5 h at pH 7 and 80 °C and an increase in melting temperature of 48.3 °C compared with the least active variant K166/R174/Y194 (inactivated after 15 min of incubation). These results, supported by structural analysis and molecular dynamics simulation studies, suggest that Lys at position 166 may stabilize the loop in which His174 is located, increasing thermal stability. View Full-Text
Keywords: NADH oxidase; cofactor regeneration; hydrogen peroxide; dehydrogenase; NAD+; extremophiles NADH oxidase; cofactor regeneration; hydrogen peroxide; dehydrogenase; NAD+; extremophiles
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Rocha-Martin, J.; Sánchez-Murcia, P.A.; López-Gallego, F.; Hidalgo, A.; Berenguer, J.; Guisan, J.M. Functional Characterization and Structural Analysis of NADH Oxidase Mutants from Thermus thermophilus HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability. Microorganisms 2019, 7, 515.

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