Structural Analysis of Aliphatic versus Aromatic Substrate Specificity in a Copper Amine Oxidase from Hansenula polymorpha

Abstract

Copper amine oxidases (CAOs) are responsible for the oxidative deamination of primary amines to their corresponding aldehydes. The CAO catalytic mechanism can be divided into two half-reactions: a reductive half-reaction, in which a primary amine substrate is oxidized to its corresponding aldehyde with the concomitant reduction of the organic cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ), and an oxidative half-reaction, in which reduced TPQ is re-oxidized with the reduction of molecular oxygen to hydrogen peroxide. The reductive half-reaction proceeds via Schiff base chemistry, in which the primary amine substrate first attacks the C5 carbonyl of TPQ, forming a series of covalent Schiff base intermediates. The X-ray crystal structures of copper amine oxidase-1 from the yeast Hansenula polymorpha (HPAO-1) in complex with ethylamine and benzylamine have been solved to resolutions of 2.18 and 2.25 Å, respectively. These structures reveal the two amine substrates bound at the back of the active site coincident with TPQ in its two-electron reduced aminoquinol form. Rearrangements of particular amino acid side chains within the substrate channel and specific protein-substrate interactions provide insight into substrate specificity in HPAO-1. These changes begin to account for this CAO’s kinetic preference for small, aliphatic amines over the aromatic amines or whole peptides preferred by some of its homologs.

Structural Snapshots from the Oxidative Half-reaction of a Copper Amine Oxidase

“…The axially bound hydrogen peroxide of these structures shows striking similarity to that seen previously in the bacterial enzyme ECAO (Fig. 5) and anaerobically ethylamine-reduced HPAO-1, where its presence with aminoquinol was proposed to be a consequence of residual O 2 in the crystallization drop leading to some initial turnover (28,43). Additionally, although H 2 O 2 is axially bound to the copper, an equatorially bound water is present at high occupancy, creating a five-coordinate copper ligation sphere.…”

Copper(II)‐Bioinspired Models for Copper Amine Oxidases: Oxidative Half‐Reaction in Water

“…There is no appreciable difference in Cu−O bond lengths ( 2 , 2.463; 3 , 2.459 Å) of both the complexes even though with different counter anions. The observed Cu−O bond distances (∼2.46 Å) is 0.24 Å lower than that in native CAOs enzymes (Cu‐O water , ∼2.7 Å), due to coulombic attraction of the anions CF 3 SO 3 − /ClO 4 − . However, the anions SO 3 CF 3 − /ClO 4 − are weakly coordinated to the copper center and labile enough to replace by a water molecule (cf.…”

“…The elongation of Cu−N bond distance in 3 due to the six‐membered chelate ring by ethylene spacer between the tertiary nitrogen atoms and pyridine rings, which leads to a less effective hybridization of d x 2 ‐ y 2 orbital of copper(II) center with p‐orbital of ligand nitrogen atoms. Interestingly, observed average Cu−N (2.0 Å) bond distances of 2 and 3 are nearly identical to the Cu‐N His bond distances of native CAOs (Cu‐N His‐456 , 2.0‐2.1 Å, Cu‐N His‐458, 1.9‐2.2 and Cu‐N His‐624, 1.9‐2.1 Å) . The N1‐Cu−N2 (92.67°) and N3‐Cu−N4 (95.23°) bond angles of 3 are higher than those in 2 (85°), which is expected as due to variation in chelate ring size.…”

“…In mammals, it removes biogenic amines from blood plasma to regulate the intracellular polyamine concentration, as the excess of polyamines leads to neurotoxicity . The active site of CAO uses copper(II) center bound at full occupancy by five ligands with distorted square‐pyramidal geometry, which includes three histidine residues His‐456 (2.0‐2.1 Å), His‐458 (1.9‐2.2 Å) and His‐624 (1.9‐2.1 Å) and two water/solvent molecules (Figure D) . Further, TPQ cofactor is formed by post‐translational modification of tyrosine residue, which is coordinated via hydrogen bonding network involving water molecules to connect the substrate binding site with the copper center.…”

A Bioinspired Organocatalytic Cascade for the Selective Oxidation of Amines under Air

“…In contrast, the biomimetic aerobic catalytic oxidation of non‐activated primary alkylamines, which are natural substrates for CuAOs, has received little attention, probably because the generated alkylimines very easily isomerize into the unstable enamine tautomers. In 2012, we reported the first biomimetic aerobic catalytic oxidation of non‐activated primary alkylamines through the synergistic combination of two redox couples: the o ‐iminoquinone organocatalyst IMQ, previously discovered from electrochemical investigations, is the substrate‐selective catalyst and the biocompatible copper salt serves as an electron transfer mediator .…”