Deciphering the role of adenosine nucleosidases (ADNs) - closing a gap in purine and cytokinin metabolism

腺苷核苷酶（Adenosine nucleosidase，ADN）是嘌呤代谢的一种酶，催化嘌呤/细胞分裂素核苷水解为相应的碱基。虽然这种酶的活性已经在几种植物中被描述，但迄今为止还没有鉴定出ADN编码基因。在我们的前期工作中，我们从苔藓小立碗藓和拟南芥中鉴定了第一个植物ADN基因（PpADN 1，-2和-3）（AtADN 1和-2）。对于PpADN 1的基因产物，我们证明了腺苷和植物激素异戊烯基腺苷（细胞分裂素）水解为相应的碱基。该项目涉及使用模式生物立碗藓和拟南芥阐明蛋白质特性和ADN在植物中的功能。将对重组ADN蛋白进行酶动力学和底物特异性的深入研究。本研究利用RT-PCR技术研究立碗藓中ADN基因转录的调控和底物诱导。ADN-GFP融合蛋白在植物中的表达将用于确定ADN的亚细胞定位。进一步的重要信息将来自拟南芥的T-DNA插入突变体，这将是腺苷和细胞分裂素代谢分析。其目的是确定在多大程度上ADN可以用于激活细胞分裂素，通过其低活性核苷转化为高活性细胞分裂素碱基。ADN的表征将缩小嘌呤/细胞分裂素互变代谢途径中的差距，预期的进展可能会打开作物改良和生物技术的应用。

Adenosine nucleosidase (ADN) is an enzyme of purine metabolism catalyzing the hydrolysis of purine/cytokinin nucleosides to the corresponding bases. Although the activity of this enzyme has been described in several plants, no ADN encoding genes were identified to date. In our preliminary work, we identified the first plant ADN genes (PpADN1, -2 and -3) from the moss Physcomitrella patens as well as from Arabidopsis thaliana (AtADN1 and -2). For the gene product of PpADN1 we proofed the hydrolysis of adenosine and the plant hormone isopentenyladenosine (cytokinin) to the corresponding bases. The project deals with the elucidation of protein properties and the function of ADN in plants using the model organisms Physcomitrella and Arabidopsis. Intensive studies on enzyme kinetics and substrate specificity will be carried out on recombinant ADN proteins. The regulation and substrate inducibility of ADN transcription will be studied in Physcomitrella using RT-PCR. Expression of ADN-GFP fusion proteins in plants will be used to determine the subcellular localisation of ADNs. Further important information will derive from T-DNA insertion mutants of Arabidopsis, which will be analysed for adenosine and cytokinin metabolism. It is aimed to determine in how far ADNs can be used to activate cytokinins by their conversion of low active ribosides to highly active cytokinin bases. Characterisation of ADNs will close a gap in the metabolic pathways of purine/cytokinin interconversion and the expected advances are likely to open applications for crop plant improvement and biotechnology.