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

Rocha-Martin, Javier ; Sánchez-Murcia, Pedro A. ; López-Gallego, Fernando (Universidad de Zaragoza) ; Hidalgo, Aurelio ; Berenguer, José ; Guisan, José M.

Resumen: 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.

Idioma: Inglés

DOI: 10.3390/microorganisms7110515

Año: 2019

Publicado en: Microorganisms 7, 11 (2019), 515 [18 pp.]
ISSN: 2076-2607

Originalmente disponible en: Texto completo de la revista

Factor impacto JCR: 4.152 (2019)

Categ. JCR: MICROBIOLOGY rank: 37 / 133 = 0.278 (2019) - Q2 - T1

Factor impacto SCIMAGO: 0.0 - Microbiology - Virology - Microbiology (medical)

Tipo y forma: Artículo (Versión definitiva)

Área (Departamento): Área Química Orgánica (Dpto. Química Orgánica)