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

含铜胺氧化酶（CuAO）是一类在所有生命领域参与调节细胞多胺水平的酶。在植物中，它们也参与几类生物碱的生物合成。这些是具有生物活性的天然化合物，是植物对食草动物的化学防御的一部分。最近，一个具有意想不到的性质的CuAO已被确定参与吡咯里西啶生物碱（PA）的生物合成。该CuAO，称为homospermidine氧化酶（HSO），催化homospermidine在两个伯氨基上的氧化，并且似乎控制中间体环化为双环吡咯里嗪环系统。分析这种PA-特异性酶在体外，在转基因植物与失活的候选基因，并通过喂养实验在植物中发现的中间体，不能解释的机制中描述的文献中的CuAO催化的氧化脱氨，即涉及形成的席夫碱与topaquinone辅因子的机制。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个CuAO。对于这一具有挑战性的任务，我们将进行经典的体外研究，以确定异源表达的CuAOs对homospermidine和相关底物的氧化。特别是，我们将专注于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饲养研究来补充。这将提供洞察的两步机制的homospermidine氧化。在公共数据库中提供的结构的基础上，结合定点诱变的酶结构的建模，应该允许阐明这些CuAO的蛋白质结构的具体特征，这些独特的生化特性。

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.