Martens Stefan, Teeri Teemu, Forkmann Gert
Center of Life and Food Science Weihenstephan, Department of Plant Science, Chair of Floriculture Crops and Horticultural Plant Breeding, Am Hochanger 4, 85350, Freising, Germany.
FEBS Lett. 2002 Nov 20;531(3):453-8. doi: 10.1016/s0014-5793(02)03583-4.
Dihydroflavonol 4-reductases (DFR) catalyze the stereospecific reduction of dihydroflavonols to the respective flavan 3,4-diols (leucoanthocyanidins) and might also be involved in the reduction of flavanones to flavan-4-ols, which are important intermediates in the 3-deoxyflavonoid pathway. Several cDNA clones encoding DFR have been isolated from different plant species. Despite the important function of these enzymes in the flavonoid pathway, attempts at heterologous expression of cDNA clones in Escherichia coli have failed so far. Here, three well known heterologous expression systems for plant-derived genes were tested to obtain the functional protein of DFR from Gerbera hybrids. Successful synthesis of an active DFR enzyme was achieved in eukaryotic cells, using either baker's yeast (Saccharomyces cerevisiae) or tobacco protoplasts (Nicotiana tabacum), transformed with expression vectors containing the open reading frame of Gerbera DFR. These expression systems provide useful and powerful tools for rapid biochemical characterization, in particular the substrate specificity, of the increasing number of cloned DFR sequences. Furthermore, this tool allows the stereospecific synthesis of (14)C-labeled leucoanthocyanidins in high quality and quantity, which is a prerequisite for detailed biochemical investigation of the less understood enzymatic reactions located downstream of DFR in anthocyanin, catechin and proanthocyanidin biosynthesis.
二氢黄酮醇4-还原酶（DFR）催化二氢黄酮醇立体特异性还原为相应的黄烷-3,4-二醇（无色花青素），也可能参与黄烷酮还原为黄烷-4-醇，这些都是3-脱氧黄酮类途径中的重要中间体。已经从不同植物物种中分离出几个编码DFR的cDNA克隆。尽管这些酶在黄酮类途径中具有重要功能，但迄今为止，在大肠杆菌中对cDNA克隆进行异源表达的尝试均告失败。在此，测试了三种著名的植物源基因异源表达系统，以从非洲菊杂种中获得DFR的功能蛋白。使用含有非洲菊DFR开放阅读框的表达载体转化的面包酵母（酿酒酵母）或烟草原生质体（烟草），在真核细胞中成功合成了活性DFR酶。这些表达系统为快速生化表征，特别是对越来越多克隆的DFR序列的底物特异性，提供了有用且强大的工具。此外，该工具能够高质量、大量地立体特异性合成（14）C标记的无色花青素，这是对花青素、儿茶素和原花青素生物合成中位于DFR下游了解较少的酶促反应进行详细生化研究的先决条件。
