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

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.This work was supported by National Institutes of Health Grant GM66569 to C.M.W, Minnesota Medical Foundation Grant 3714-9221-06, Office of the Dean of the Graduate School of the University of Minnesota Grant 21087, a 3M Science and Technology fellowship to V.J.K., a University of Minnesota UROP grant to C.J.S., and a Minnesota Partnership for Biotechnology and Medical Genomics Grant SPAP-05-0013-P-FY06 to C.M.W. It was sponsored by the Undergraduate Research Opportunities Program