Identification of Two Imidazole Binding Sites and Key Residues for Substrate Specificity in Human Primary Amine Oxidase AOC3

Abstract

Human membrane primary amine oxidase (hAOC3; also known as vascular adhesion protein-1, VAP-1) is expressed upon inflammation in most tissues, where its enzymatic activity plays a crucial role in leukocyte trafficking. We have determined two new structures of a soluble, proteolytically cleaved form of hAOC3 (sAOC3), which was extracted from human plasma. In the 2.6 Å sAOC3 structure, an imidazole molecule is hydrogen bonded to the topaquinone (TPQ) cofactor, which is in an inactive on-copper conformation, while in the 2.95 Å structure, an imidazole molecule is covalently bound to the active off-copper conformation of TPQ. A second imidazole bound by Tyr394 and Thr212 was identified in the substrate channel. We furthermore demonstrated that imidazole has an inhibitory role at high concentrations used in crystallization. A triple mutant (Met211Val/Tyr394Asn/Leu469Gly) of hAOC3 was previously reported to change substrate preferences toward those of hAOC2, another human copper-containing monoamine oxidase. We now mutated these three residues and Thr212 individually to study their distinct role in the substrate specificity of hAOC3. Using enzyme activity assays, the effect of the four single mutations was tested with four different substrates (methylamine, benzylamine, 2-phenylethylamine, and p-tyramine), and their binding modes were predicted by docking studies. As a result, Met211 and Leu469 were shown to be key residues for substrate specificity. The native structures of sAOC3 and the mutational data presented in this study will aid the design of hAOC3 specific inhibitors.

Novel Pyridazinone Inhibitors for Vascular Adhesion Protein-1 (VAP-1): Old Target–New Inhibition Mode

“…These novel inhibitors block the entrance and hinder any ligand of entering the active site (Figure 2). The binding site is distinct but close to the position that was named as the secondary imidazole binding site in our previously published VAP-1 structures 18 .…”

“…In the inhibitor bound structures, Phe173 has taken an alternative conformation to allow passage and create additional space for the inhibitors to bind. Compared to the native (PDB code 1US1 13 ) and imidazole bound structures (PDB codes 2Y73 and 2Y74 18 ), the phenyl ring of Phe389 interacts with all inhibitors and it is slightly rotated to make more favorable interactions to molecules 6 and 7 . Root-mean-square-differences (rmsd) after superposition of 6 , 7 , and 13 bound structures to the native structure are 0.40 Å (1339 atoms), 0.28 Å (1282 atoms), and 0.32 Å (1299 atoms), respectively.…”

“…However, Thr212 in all A chains is flipped 180 degrees and does not interact with the inhibitors (Figure 2B, C, E), but instead forms a hydrogen bond with Arg216. Similarly, in the human VAP-1 imidazole complex structure (PDB code 2Y73 18 ) Thr212 interacted with Arg216 in one chain, but in the other chain a water molecule mediated its interaction with the imidazole. In both 7 and 13 bound complexes, the oxygen atom of the pyridazinone interacts with a water molecule (Figure 2C, D, E).…”

“…New structures of human VAP-1 in complex with imidazole molecules, presented a secondary imidazole binding site in the active site channel 18 . This raised the idea that the active site channel may provide a new target area for drug design for human VAP-1 inhibitors, which has thereafter been discussed by Bonaiuto and coworkers in relation to synthetic polyamines 19 .…”

Multivalent Interactions of Human Primary Amine Oxidase with the V and C22 Domains of Sialic Acid-Binding Immunoglobulin-Like Lectin-9 Regulate Its Binding and Amine Oxidase Activity

“…Finally, the protein was purified by gel filtration on a Superdex 200 10/300 GL (GE Healthcare) column in 20 mM HEPES pH 7.4, 150 mM NaCl. hAOC3 production and purification was carried out exactly as previously described [36]. …”