Evolution and functional classification of mammalian copper amine oxidases

Mammalian copper-containing amine oxidases (CAOs), encoded by four genes (AOC1-4) and catalyzing the oxidation of primary amines to aldehydes, regulate many biological processes and are linked to various diseases including inflammatory conditions and histamine intolerance. Despite the known differences in their substrate preferences, CAOs are currently classified based on their preference for either primary monoamines (EC 1.4.3.21) or diamines (EC 1.4.3.22). Here, we present the first extensive phylogenetic study of CAOs that, combined with structural analyses of the CAO active sites, provides in-depth knowledge of their relationships and guidelines for classification of mammalian CAOs into AOC1-4 sub-families. The phylogenetic results show that CAOs can be classified based on two residues, X1 and X2, from the active site motif: T/S-X1-X2-N-Y-D. Residue X2 discriminates among the AOC1 (Tyr), AOC2 (Gly), and AOC3/AOC4 (Leu) proteins, while residue X1 further classifies the AOC3 (Leu) and AOC4 (Met) proteins that so far have been poorly identified and annotated. Residues X1 and X2 conserved within each sub-family and located in the catalytic site seem to be the key determinants for the unique substrate preference of each CAO sub-family. Furthermore, one residue located at 10 Å distance from the catalytic site is different between the sub-families but highly conserved within each sub-family (Asp in AOC1, His in AOC2, Thr in AOC3 and Asn in AOC4) and likely contributes to substrate selectivity. Altogether, our results will benefit the design of new sub-family specific inhibitors and the design of in vitro tests to detect individual CAO levels for diagnostic purposes.