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

含铜胺氧化酶（CuAOs）是一类特性良好的酶，在生命的所有领域都参与调节细胞多胺水平。在植物中，它们还参与几种生物碱的生物合成。这些是具有生物活性的天然化合物，是植物抵御食草动物的化学防御的一部分。最近，一种具有意想不到性质的CuAO被发现参与了吡咯利西啶生物碱（PA）的生物合成。这种CuAO称为同亚精胺氧化酶（HSO），催化同亚精胺在两个一级氨基上的氧化，并似乎控制中间体到双环吡咯利西啶环系统的环化。对这种pa特异性酶的体外、候选基因失活的转基因植物以及植物取食实验的分析，揭示了无法用文献中描述的cuao催化氧化脱氨机制来解释的中间体，即与托喹酮辅因子形成希夫碱的机制。因此，我们假设某些cuao可能使用不同的机制来氧化伯胺，我们将在这方面分析几种cuao。为了完成这项具有挑战性的任务，我们将进行经典的体外研究，以表征异源表达的cuao对同亚精胺和相关底物的氧化作用。特别是，我们将重点关注形成的产品和中间体。这些研究将通过使用具有失活候选基因的转基因植物的摄食研究加以补充。这将有助于深入了解同亚精胺氧化的两步机制。基于公共数据库中可用的结构对酶结构进行建模，并结合位点定向诱变，应该能够阐明这些具有独特生化特性的CuAOs的蛋白质结构的具体特征。

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.