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

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 co...

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Authors: Rocha-Martin, Javier, Sánchez-Murcia, Pedro A., López-Gallego, Fernando, Hidalgo, Aurelio, Berenguer, José, Guisan, José M.
Format: article
Status:Published version
Publication Date:2019
Country:España
Institution:Universidad de Zaragoza
Repository:Zaguán. Repositorio Digital de la Universidad de Zaragoza
OAI Identifier:oai:zaguan.unizar.es:86244
Online Access:http://zaguan.unizar.es/record/86244
Access Level:Open access
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spelling 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 thermostabilityRocha-Martin, JavierSánchez-Murcia, Pedro A.López-Gallego, FernandoHidalgo, AurelioBerenguer, JoséGuisan, José M.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.2019info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttp://zaguan.unizar.es/record/86244reponame:Zaguán. Repositorio Digital de la Universidad de Zaragozainstname:Universidad de ZaragozaInglésinfo:eu-repo/semantics/openAccessoai:zaguan.unizar.es:862442026-05-29T13:59:51Z
dc.title.none.fl_str_mv 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
title 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
spellingShingle 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
title_short 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
title_full 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
title_fullStr 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
title_full_unstemmed 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
title_sort 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
dc.creator.none.fl_str_mv Rocha-Martin, Javier
Sánchez-Murcia, Pedro A.
López-Gallego, Fernando
Hidalgo, Aurelio
Berenguer, José
Guisan, José M.
author Rocha-Martin, Javier
author_facet Rocha-Martin, Javier
Sánchez-Murcia, Pedro A.
López-Gallego, Fernando
Hidalgo, Aurelio
Berenguer, José
Guisan, José M.
author_role author
author2 Sánchez-Murcia, Pedro A.
López-Gallego, Fernando
Hidalgo, Aurelio
Berenguer, José
Guisan, José M.
author2_role author
author
author
author
author
description 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.
publishDate 2019
dc.date.none.fl_str_mv 2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
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dc.identifier.none.fl_str_mv http://zaguan.unizar.es/record/86244
url http://zaguan.unizar.es/record/86244
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv
publisher.none.fl_str_mv
dc.source.none.fl_str_mv reponame:Zaguán. Repositorio Digital de la Universidad de Zaragoza
instname:Universidad de Zaragoza
instname_str Universidad de Zaragoza
reponame_str Zaguán. Repositorio Digital de la Universidad de Zaragoza
collection Zaguán. Repositorio Digital de la Universidad de Zaragoza
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