Catalytic Mechanism of the Quinoenzyme Amine Oxidase from Escherichia coli:  Exploring the Reductive Half-Reaction^,

Abstract

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.

Exploring Molecular Oxygen Pathways in Hansenula polymorpha Copper-containing Amine Oxidase

“…4, d-f) and relates well to the fact that HPAO is the only characterized CAO with a specific preference for small aliphatic primary amines (ethylamine Ͼ methylamine Ͼ Ͼ benzylamine) (51,56). Modeling of methylamine and ethylamine Schiff base intermediates from the reductive half-reaction did not interfere with the proposed O 2 pathway from the amine channel, although the Schiff base with benzylamine did (54,57). However, because benzylamine is a poor substrate for the enzyme, with a 100-fold decrease in k cat compared with ethylamine, it is not thought to be a physiologically relevant substrate (58).…”

“…the aromatic portion of its preferred aromatic monoamine substrates and has a stabilizing and orienting effect (54). Because no side-chain movement is involved in the binding of Xe2 in HPAO, Trp-156 does not seem to act as a gate per se, being already in the open conformation (Fig.…”

Structure of a quinohemoprotein amine dehydrogenase with an uncommon redox cofactor and highly unusual crosslinking

“…Conversion of the substrate Schiff base to the product Schiff base, during which a carbanion intermediate is formed and the cofactor becomes reduced, is catalyzed by proton abstraction by a catalytic base on the enzyme. This base has been identified as an aspartate residue in CAO (28) and is likely to be an aspartate in MADH (29,30). In QHNDH, the only side chain present in the active site that can act as the catalytic base is Asp-33␥, the others being hydrophobic or aromatic.…”

Synthesis and evaluation of resins bearing substrate-like inhibitor functions for capturing copper amine oxidases

“…(E)-2,6-Dimethoxy-4-(4-hydroxy-2-butenyloxy)benzylamine hydrochloride (16). A suspension of LiAlH 4 (1.01 g, 26.6 mmol) in dry Et 2 O (160ml) was cooled to 0 1C under N 2 , treated dropwise with a solution of (E)-2,6-dimethoxy-4-[(2-tetrahydropyranyloxy)-2-butenyloxy]benzonitrile (12) (Supplementary Information) (4.37 g, 13.1 mmol) in dry Et 2 O (100 ml) at reflux for 3 h 30 min and left overnight at room temperature until disappearance of the infrared (IR) nitrile band at 2200 cm À1 .…”

“…16 Our contribution in this field mainly focuses on the synthesis of the first substrate-like inhibitors classified as highly selective, partially reversible for benzylamine oxidase (BAO) having a benzylamine structure, 17 highly selective, fully reversible for BAO having a 4-aminomethylpyridine structure, 18 and highly active, nonselective for BAO and diamine oxidase (DAO) having a 4-aminomethylpyridine structure. 18 As CAOs are characterized by a moderate substrate selectivity, making their biological role rather complex and difficult to elucidate, further research is needed.…”