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

含铜胺氧化酶(CuAO)是一类广泛存在于生命各个领域的酶，参与调节细胞内多胺水平。在植物中，它们还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，是植物对抗食草动物的化学防御的一部分。最近，一种具有意想不到的性质的CuAO被发现，它参与了吡咯里西丁生物碱(PA)的生物合成。这种CuAO被称为高精胺氧化酶(HSO)，它催化高精胺在两个伯氨基上的氧化，似乎控制着中间体到双环吡咯里西啶环系的环化。在体外、在含有失活候选基因的转基因植物中对这种PA专一性酶的分析，以及在植物中的饲养实验，发现了不能用文献中描述的CuAO催化的氧化脱氨反应的机制来解释的中间产物，即涉及与TOPQUEON辅因子形成席夫碱的机制。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个CuAO。为了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对高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含有失活候选基因的转基因植物进行喂养研究来补充。这将为研究高香草胺氧化的两步反应机制提供依据。在公共数据库中现有结构的基础上对酶结构进行建模，结合定点突变，应该能够阐明这些具有这种独特生化特性的CuAO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.