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

Abstract

Copper amine oxidases (CAOs) are enzymes that carry out oxidative deamination of primary amines , generating their corresponding aldehydes. The catalytic mechanism (Figure 1) is ping-pong and can be divided into two half-reactions: a reductive half reaction, in which an active site topa quinone (TPQ) cofactor is reduced with the release of product aldehyde, and an oxidative half-reaction, in which reduced TPQ is reoxidized with the concomitant reduction of O2 to H2O2. The reductive half-reaction proceeds via Schiff base chemistry, in which substrate amine first attacks the C5 carbonyl of TPQ, forming a series of Schiff base intermediates.

In order to explore the structural factors which influence substrate specificity in HPAO-1, its crystals were anaerobically reduced with methylamine (MeAm), ethylamine (EtAm), and benzylamine (BeAm) before flash-freezing, and their structures have been solved by X-ray crystallography. Comparison of the structures of HPAO-1 in complex with the three different resulting aldehyde products as compared to that of native HPAO-1 provides insight into specific substrate-protein interactions involving residues located within the substrate channel.