Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.

In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site...

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Autores principales:	Martin Mahro, Natércia F Brás, Nuno M F S A Cerqueira, Christian Teutloff, Catarina Coelho, Maria João Romão, Silke Leimkühler
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/126216c92ec64e3a972223d13d5e6cbd
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spelling	oai:doaj.org-article:126216c92ec64e3a972223d13d5e6cbd2021-11-18T08:41:50ZIdentification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.1932-620310.1371/journal.pone.0082285https://doaj.org/article/126216c92ec64e3a972223d13d5e6cbd2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24358164/?tool=EBIhttps://doaj.org/toc/1932-6203In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.Martin MahroNatércia F BrásNuno M F S A CerqueiraChristian TeutloffCatarina CoelhoMaria João RomãoSilke LeimkühlerPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e82285 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Martin Mahro Natércia F Brás Nuno M F S A Cerqueira Christian Teutloff Catarina Coelho Maria João Romão Silke Leimkühler Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
description	In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.
format	article
author	Martin Mahro Natércia F Brás Nuno M F S A Cerqueira Christian Teutloff Catarina Coelho Maria João Romão Silke Leimkühler
author_facet	Martin Mahro Natércia F Brás Nuno M F S A Cerqueira Christian Teutloff Catarina Coelho Maria João Romão Silke Leimkühler
author_sort	Martin Mahro
title	Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
title_short	Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
title_full	Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
title_fullStr	Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
title_full_unstemmed	Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
title_sort	identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/126216c92ec64e3a972223d13d5e6cbd
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Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity.

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