Zhu Yue, Peng Qing-Zhong, Li Ke-Gang, Xie De-Yu
Hunan Provincial Key Laboratory of Plant Resources Conservation and Utilization, College of Biology and Environmental Sciences, Jishou University, No. 120 Ren Min Nan Lu, Jishou City, 416000, Hunan Province, People's Republic of China.
Planta. 2014 Aug;240(2):381-98. doi: 10.1007/s00425-014-2094-2. Epub 2014 Jun 1.
Anthocyanidin reductase (ANR) is an NADPH-/NADH-dependent enzyme that transfers two hydrides to anthocyanidins to produce three types of isomeric flavan-3-ols. This reductase forms the ANR pathway toward the biosynthesis of proanthocyanidins (PAs, which are also called condensed tannins). Here, we report cloning and functional characterization of an ANR (called VbANR) homolog from the leaves of Vitis bellula, a newly developed grape crop in southern China. The open reading frame (ORF) of VbANR is 1,017 bp in length and encodes 339 amino acids. A phylogenetic analysis and an alignment using 17 sequences revealed that VbANR is approximately 99.9 % identical to the ANR homolog from Vitis vinifera. The VbANR ORF is fused to the Trx gene containing a His-tag in the pET32a(+) vector to obtain a pET32a(+)-VbANR construct for expressing the recombinant VbANR. In vitro enzyme assays show that VbANR converts cyanidin, delphinidin, and pelargonidin to their corresponding flavan-3-ols. Enzymatic products include 2S,3R-trans- and 2R,3R-cis-flavan-3-ols isomers, such as (-)-catechin and (-)-epicatechin. In addition, the third compound that is observed from the enzymatic products is most likely a 2S,3S-cis-flavan-3-ol. To analyze the kinetics and optimize pH and temperature values, a UV spectrometry method was developed to quantify the concentrations of total enzymatic products. The optimum pH and temperature values are 4.0 and 40 °C, respectively. The K m , K cat, V max, and K cat/K m values for pelargonidin and delphinidin were similar. In comparison, VbANR exhibits a slightly lower affinity to cyanidin. VbANR uses both NADPH and NADH but prefers to employ NADPH. GFP fusion and confocal microscopy analyses revealed the cytosolic localization of VbANR. The overexpression of VbANR in ban mutants reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate that VbANR forms the ANR pathway, leading to the formation of three types of isomeric flavan-3-ols and PAs in the leaves of V. bellula.
花青素还原酶（ANR）是一种依赖NADPH/NADH的酶，它将两个氢化物转移到花青素上，生成三种类型的异构黄烷-3-醇。这种还原酶构成了原花青素（PAs，也称为缩合单宁）生物合成的ANR途径。在此，我们报告了从小叶葡萄（中国南方新培育的葡萄品种）叶片中克隆得到的一种ANR（称为VbANR）同源物及其功能特性。VbANR的开放阅读框（ORF）长度为1017 bp，编码339个氨基酸。通过对17个序列进行系统发育分析和比对发现，VbANR与酿酒葡萄中的ANR同源物相似度约为99.9%。将VbANR的ORF与含有His标签的Trx基因在pET32a(+)载体中融合，以获得用于表达重组VbANR的pET32a(+)-VbANR构建体。体外酶活性分析表明，VbANR可将矢车菊素、飞燕草素和天竺葵素转化为相应的黄烷-3-醇。酶促产物包括2S,3R-反式和2R,3R-顺式黄烷-3-醇异构体，如（-）-儿茶素和（-）-表儿茶素。此外，从酶促产物中观察到的第三种化合物很可能是2S,3S-顺式黄烷-3-醇。为了分析动力学并优化pH和温度值，开发了一种紫外光谱法来定量总酶促产物的浓度。最佳pH和温度值分别为4.0和40℃。天竺葵素和飞燕草素的K m、K cat、V max和K cat/K m值相似。相比之下，VbANR对矢车菊素的亲和力略低。VbANR既能利用NADPH也能利用NADH，但更倾向于使用NADPH。绿色荧光蛋白（GFP）融合和共聚焦显微镜分析显示VbANR定位于细胞质。在ban突变体中过表达VbANR可重建种皮中原花青素的生物合成途径。这些数据表明，VbANR构成了ANR途径，导致小叶葡萄叶片中形成三种类型的异构黄烷-3-醇和原花青素。
