Copper-containing amine oxidases – Characterization of a class of enzymes involved in alkaloid biosynthesis and beyond

• Applicant: Professor Dr. Dietrich Ober
• Subject Area: Organismic Interactions, Chemical Ecology and Microbiomes of Plant Systems
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 534215564

Copper-containing amine oxidases (CuAOs) are a well-characterised class of enzymes in all domains of life involved in regulating cellular polyamine levels. In plants, they are also involved in the biosynthesis of several classes of alkaloids. These are bioactive natural compounds that are part of the plant’s chemical defence against herbivores. Recently, a CuAO with unexpected properties has been identified that is involved in pyrrolizidine alkaloid (PA) biosynthesis. This CuAO, called homospermidine oxidase (HSO), catalyses the oxidation of homospermidine at both primary amino groups and seems to control the cyclisation of the intermediate to the bicyclic pyrrolizidine ring system. Analysis of this PA-specific enzyme in vitro, in transgenic plants with inactivated candidate genes, and by feeding experiments in planta revealed intermediates that cannot be explained by the mechanism described in the literature for CuAO-catalyzed oxidative deaminations, namely a mechanism involving the formation of a Schiff base with the topaquinone cofactor. Therefore, we postulate that certain CuAOs may use a different mechanism to oxidise primary amines and we will analyse several CuAOs in this respect. For this challenging task, we will perform classical in vitro studies to characterise the oxidation of homospermidine and related substrates by heterologously expressed CuAOs. In particular, we will focus on the products and intermediates formed. These studies will be complemented by feeding studies using transgenic plants with inactivated candidate genes. This will provide insight into the two-step mechanism of homospermidine oxidation. Modelling of the enzyme structure on the basis of structures available in the public databases, combined with site-directed mutagenesis, should allow to elucidate the specific features of the protein structures of these CuAOs with such unique biochemical properties.

• DFG Programme: Research Grants