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

PLoS One. 2013 Dec 16;8(12):e82285. doi: 10.1371/journal.pone.0082285. eCollection 2013.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aldehyde Oxidoreductases / genetics
  • Aldehyde Oxidoreductases / metabolism*
  • Amino Acid Sequence
  • Animals
  • Mice
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Substrate Specificity


  • Aldehyde Oxidoreductases
  • Aox3 protein, mouse

Grant support

This work was supported by the Cluster of Excellence “Unifying Concepts in Catalysis” (to C.T. and S.L.) coordinated by the Technische Universität Berlin and funded by the Deutsche Forschungsgemeinschaft. The exchange of researchers among laboratories involved in the work was funded by the DAAD-GRICES program (to MJ Romão and S Leimkühler). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.