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

含铜胺氧化酶 â 涉及生物碱生物合成等的一类酶的表征

• 批准号: 534215564
• 负责人: Professor Dr. Dietrich Ober
• 金额: --
• 依托单位: Botanisches Institut und Botanischer Garten
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/534215564?language=en
• 关键词: Copper; containing; amine; oxidases; Characterization

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.

含铜的胺氧化物（Cuaos）是在调节细胞多胺水平的所有生命领域中均匀的一类酶。在植物中，它们还参与了几类生物碱的生物合成。这些是生物活性的天然化合物，是该植物对草食动物的化学防御的一部分。最近，已经鉴定出具有意外特性的CUAO，该Cuao与吡咯烷碱生物碱（PA）生物合成有关。这种称为同性恋氧化物（HSO）的CuAO将同倍氨基丁烷在两个初级氨基组中氧化催化，并似乎控制了中间体对生物吡咯烷环系统的旋风。在具有失活候选基因的转基因植物中，对这种PA特异性酶的分析，以及通过在植物学中进行的喂养实验，揭示了中间体无法通过文献中描述的Cuao催化氧化脱氨酸的机制来解释这些中间体，即涉及与Topal cofactor cofactor cofactor的机制。因此，我们假设某些Cuaos可能会使用不同的机制氧化原代胺，我们将在这方面分析几种Cuaos。对于这项挑战任务，我们将进行经典的体外研究，以表征异源性表达的Cuaos对同伴和相关底物的氧化。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灭活候选基因的转基因植物的喂养研究来完成。这将提供有关同倍氨酸氧化的两步机制的洞察力。基于公共数据库中可用的结构进行建模，结合定向诱变，应允许具有如此独特的生化特性的这些Cuaos的蛋白质结构的特定特征。