Direct comparison of the four aldehyde oxidase enzymes present in mouse gives insight into their substrate specificities

Abstract

Mammalian aldehyde oxidases (AOXs) are molybdo-flavoenzymes which are present in many tissues in various mammalian species, including humans and rodents. Different species contain a different number of AOX isoforms. In particular, the reasons why mammals other than humans express a multiplicity of tissue-specific AOX enzymes is unknown. In mouse, the isoforms mAOX1, mAOX3, mAOX4 and mAOX2 are present. We previously established a codon-optimized heterologous expression systems for the mAOX1-4 isoforms in Escherichia coli that gives yield to sufficient amounts of active protein for kinetic characterizations and sets the basis in this study for site-directed mutagenesis and structure-function studies. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes on a larger number of substrates has never been performed. Here, thirty different structurally related aromatic, aliphatic and N-heterocyclic compounds were used as substrates, and the kinetic parameters of all four mAOX enzymes were directly compared. The results show that especially mAOX4 displays a higher substrate selectivity, while no major differences between mAOX1, mAOX2 and mAOX3 were identified. Generally, mAOX1 was the enzyme with the highest catalytic turnover for most substrates. To understand the factors that contribute to the substrate specificity of mAOX4, site-directed mutagenesis was applied to substitute amino acids in the substrate-binding funnel by the ones present in mAOX1, mAOX3, and mAOX2. An increase in activity was obtained by the amino acid exchange M1088V in the active site identified to be specific for mAOX4, to the amino acid identified in mAOX3.

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“…Association region 19 (1,326,444-1,374,494 bp) linked to 4-hydroxy acetophenone (UR), acetophenone (UR), and benzyl acetate (UR) contains a candidate gene encoding a "GLOX Aldehyde oxidase." An aldehyde oxidase is in some cases responsible for the oxidation of phenylacetaldehyde to phenylacetate, both of which are part of the L-phenylalanine degradation pathway (Kücükgöze and Leimkühler, 2018). Association zone 20 (1,380,802-1,510,054 bp) linked to 4hydroxy acetophenone (UR), acetophenone (UR), benzyl acetate (UR), and cinnamaldehyde (R) contains six candidate genes, four of which code for an Aldo-keto reductase family 4 member C9 and two for an Aldo-keto reductase family 4 member C8 (Figure 8; Supplementary Figure 6).…”

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“…In mice, only AOX2 and AOX3 can metabolize MNAM. Mouse AOX2 has much higher catalytic efficiency for MNAM (k cat /K M = 5.2 min −1 µM −1 ) than AOX3 (k cat /K M = 0.7 min −1 µM −1 ) [38]. The expression of AOX2 is highly restricted to the Bowman's gland in the nasal cavity, while AOX3 is expressed in the liver, lung, oviduct and testis [39].…”