Amine oxidases (AOs) catalyze the deamination of primary amines to their corresponding aldehydes producing ammonia and hydrogen peroxide (Eq. 1).
RCH2NH3+ + O2 + H2O → RCHO + NH4 + + H2O2 (1)
Amine oxidases can be divided into two groups based on the cofactors they utilize: quinone and copper-containing amine oxidases (CuAOs) and flavin-dependent monoamine oxidases (MAOs). MAOs are located exclusively in the outer mitochondrial membrane of almost all cell types and can oxidize primary, secondary, and tertiary amines either by a concerted covalent catalysis or by a single electron-transfer mechanism, both requiring FAD as a cofactor . Quinone copper-containing amine oxidases generally oxidize primary amines and can be subdivided based on the cofactor present in the active site. The first group contains 2,4,5-trihydroxyphenylalanine quinone (TPQ) which is formed in a self-processing post-translational modification of a conserved tyrosine within the sequence Ser/Thr - X aa - X aa - Asn – Tyr (TPQ) - Asp/Glu - Tyr/Asn. Molecular oxygen and copper are required in order for this modification to occur. The second class of quinone copper-containing amine oxidases use lysyl tyrosylquinone as their cofactor and are referred to as lysyl oxidases. Lysyl oxidases are involved in connective tissue maturation through the deamination of peptidyl lysine side chains that initiate lysine residue cross-linking in collagen and elastin.
Copper-containing amine oxidases can be found throughout nature and have been purified from many sources including plants, mammals, and microorganisms. In microorganisms, CuAOs play a nutritional role allowing primary amines to be utilized as the sole source of carbon and nitrogen. In higher organisms the physiological roles of CuAOs are not yet fully clear. In plants, it is theorized that amine oxidases aid in the biosynthesis of hormones, cell walls, and alkaloids. In mammals, function seems to be tissue specific involving physiological response to injury, apoptosis, cell growth, signaling, and detoxification. Four types of TPQ copper amine oxidases have been described from mammalian sources: plasma amine oxidase, diamine oxidase (DAO), retinal amine oxidase, and semicarbazide-sensitive amine oxidase (SSAO). SSAO has multiple functions depending on its location, including glucose homeostasis, lymphocyte adhesion, and adipocyte maturation. It was recently discovered that human kidney diamine oxidase (KDAO) has the highest substrate specificity with 1-methylhistamine and histamine, indicating that this enzyme could play an integral but as yet undetermined role in histamine metabolism.
Five CuAO structures have been experimentally determined: Escherichia coli amine oxidase (ECAO), Pisum sativum amine oxidase (PSAO), Pichia pastoris lysyl oxidase (PPLO), Hansenula polymorpha amine oxidase (HPAO, recently reclassified as Pichia angusta(PAAO)), and Arthrobacter globiformis amine oxidase (AGAO). These CuAOs are dimers with monomeric molecular masses ranging from 70 to 90 kDa, and with each monomer containing a single active site composed of TPQ and a Cu(II) atom. Each monomer has a pair of β-hairpin arms, which extend from one subunit across the face of the other subunit. One of these arms partially defines the entrance to the active site channel in the other subunit. The residues of this arm vary among the CuAOs and may play a role in substrate recognition. Differences in amino acid composition in this region introduce unique characteristics to a given CuAO in terms of the electrostatic properties and dimensions of the active site channel, as well as the degree of substrate accessibility to TPQ.
Copper-containing amine oxidases oxidize primary amines through a ping-pong mechanism (Scheme 1). The key step in catalysis is the conversion of the initial “substrate Schiff base”, a quinoneimine, to the “product Schiff base”, a quinolaldimine (B→C). This conversion is facilitated by a conserved aspartate acting as a general base assisting proton abstraction from the alpha carbon of the substrate. Subsequently the aldehyde product is released through hydrolysis, yielding Cu(II)-aminoresorcinol in equilibrium with Cu(I)-semiquinone (D↔E). In the presence of O2, oxidation to an iminoquinone species occurs, producing H2O2. This species is then hydrolyzed, liberating NH4+ and the resting cofactor (F→A). In addition, NH4+ may be released by a transimination reaction between the iminoquinine and substrate, thereby forming the “substrate Schiff base” (F→B ). It is possible that there are slight variations among CuAOs with respect to the mechanism for reoxidizing the reduced quinone species. In a proposed alternate mechanism for the reoxidation of TPQ red in HPAO, the reduction of Cu(II) to Cu(I) is not required.
In the lab we purify AGAO, PPLO, PSAO, hDAO, PAAO, and BPAO.
We are investigating the structural and biochemical foundations for the diverse biological roles of amine oxidases by continuing crystallographic, spectroscopic, and reactivity studies of selected amine oxidases. Particular attention is being paid to studies of the human vascular adhesion protein (hVAP-1, also known as semicarbazide-sensitive amine oxidase, SSAO), and to human diamine oxidase (hDAO). The interactions of selected substrates and inhibitors with multiple amine oxidases is being explored to define factors that control substrate specificity and inhibitor selectivity.
Mechanistic studies of CuAOs aimed at two critical issues are being pursued: (i) the means of electronic activation of the precursor tyrosine and the mechanism of the O2 dependent steps of TPQ biogenesis; (ii) the mechanism of oxygen activation and reoxidation of the substrate-reduced amine oxidase.