Vetter H P, Mangold U, Schröder G, Marner F J, Werck-Reichhart D, Schröder J
Institut für Biologie II, Lehrstuhl für Biochemie der Pflanzen, Universität Freiburg, Schänzlestrasse 1, D-7800 Freiburg, Federal Republic of Germany.
Plant Physiol. 1992 Oct;100(2):998-1007. doi: 10.1104/pp.100.2.998.
We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 +/- 0.5 and 6 +/- 1 mug/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus.
我们筛选了来自长春花（Catharanthus roseus）细胞培养物的cDNA文库，这些细胞培养物经诱导用于吲哚生物碱合成，并通过差异杂交、杂交mRNA的大小以及许多P-450家族中保守的氨基酸基序的存在，选择了诱导型细胞色素P-450（P-450）蛋白的克隆。对满足这些标准的四个cDNA进行了详细分析。它们被分为两类（pCros1、pCros2），代表一个新的P-450家族CYP72的两个密切相关的基因。针对在大肠杆菌中过表达的cDNA融合蛋白制备的抗血清在长春花中识别出一条56 kD的蛋白条带。蛋白质印迹定量显示，在未诱导和诱导细胞的膜中，它分别占蛋白质的1.5±0.5和6±1 μg/mg，并且对总P-450含量的分析表明，cDNA编码的蛋白是主要的P-450蛋白之一。吲哚生物碱的合成途径包含两种已知的P-450酶，香叶醇-10-羟化酶（GE10H）和橙花醇-10-羟化酶（NE10H）。克隆的P-450蛋白和GE10H活性的诱导动力学相似，但NE10H的不同。用其他植物的膜进行的蛋白质印迹表明，P-450 CYP72对长春花和其他具有GE10H活性的植物具有特异性。讨论了将CYP72初步指定为GE10H的情况。将cDNA重新克隆用于在酿酒酵母中表达，并通过蛋白质印迹证明了该蛋白的存在。对GE10H的检测未能检测到酶活性，对长春花中存在的NE10H和其他P-450酶也得到了相同的阴性结果。
