Identification of Crucial Amino Acids in Mouse Aldehyde Oxidase 3 That Determine Substrate Specificity

Abstract

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.

Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms

“…Here, a protein was obtained in which all cofactors were inserted; however, the protein was shown to be inactive. Since the glycine is located next to the catalytically essential glutamate, it might therefore negatively affect the first step of catalysis, which is the proton abstraction of the Mo-OH group by the deprotonated glutamate (E1270), which acts as an active-site base at the pH optimum of 8.0 of the enzyme (Coelho et al, 2012;Mahro et al, 2013). The generated Mo-O -group then performs a nucleophilic attack on the carbon atom of the substrate in the second step of the reaction mechanism.…”

Systematic exploration of predicted destabilizing nonsynonymous single nucleotide polymorphisms (nsSNPs) of human aldehyde oxidase: A Bio‐informatics study

“…Another interesting nsSNP at the active site is Q776V, but so far, no kinetic data are available. Q776 residue is a highly conserved residue among XO and AOX family members, that makes an H bond with the Mo apical oxygen ligand (2.9 Å) (Figure A and B). The substitution of Gln by a Val residue might influence the catalytic activity of the enzyme but not the structure of the protein, and according to our in silico results, this nucleotide change gives rise to a stabilizing nsSNP, but with no relevant effect on the protein structure (ΔΔG = 0 kcal/mol).…”

“…Another interesting nsSNP at the active site is Q776V, but so far, no kinetic data are available. Q776 residue is a highly conserved residue among XO and AOX family members, 9,47,48 that makes an H bond with the Mo apical oxygen ligand (2.9 Å) ( Figure 5A and B). Domain III includes also the noncompetitive inhibition site, where thioridazine, and likely other phenothiazine family members, binds.…”

Species-Specific Involvement of Aldehyde Oxidase and Xanthine Oxidase in the Metabolism of the Pyrimidine-Containing mGlu5-Negative Allosteric Modulator VU0424238 (Auglurant)

“…Humans, monkeys, and guinea pigs, for example, express only a single liver isoform, aldehyde oxidase 1 (AOX1), whereas mice and rats express two: AOX1 and AOX3 (Terao et al, 2016). Furthermore, the AOX3 isoform is present in higher quantities than AOX1 in mouse hepatic tissue, and computational and site-directed mutagenesis studies suggest differences in the substrate specificities of these two isoforms (Coelho et al, 2012;Mahro et al, 2013;Cerqueira et al, 2015;Terao et al, 2016). Recently, a direct comparison of mouse AOX1 and AOX3 revealed substantial differences in the kinetic parameters of six substrates between the two isoforms (Kucukgoze et al, 2017).…”