Martin G E, Timko M P, Wilks H M
Krebs Institute for Biomolecular Research and Robert Hill Institute for Photosynthesis, Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, U.K.
Biochem J. 1997 Jul 1;325 ( Pt 1)(Pt 1):139-45. doi: 10.1042/bj3250139.
protochlorophyllide oxidoreductase (POR) catalyses the light-dependent reduction of protochlorophyllide to chlorophyllide, a key reaction in the chlorophyll biosynthetic pathway. To facilitate structure-function studies, POR from pea (Pisum sativum L.) has been overexpressed in Escherichia coli as a fusion with maltose-binding protein (MBP) at 5-10% of the total soluble cell protein. The fusion protein (MBP-POR) has been purified to greater than 90% homogeneity by a two-step affinity-purification procedure. This represents the first successful overexpression and purification of a plant POR. MBP-POR was found to be active, and the kinetic properties were determined using a continuous assay in which the rate of chlorophyllide formation was measured. The Vmax was 20.6+/-0.9 nmol.min-1.mg-1 and the Km values for NADPH and protochlorophyllide were 8.7+/-1.9 microM and 0.27+/-0.04 microM respectively. These results represent the first determination of the kinetic properties of a pure POR and the first report on the kinetics of POR from a dicotyledenous plant. The experiments described here demonstrate that the enzyme is not a 'suicide' enzyme, and the only components required for catalysis are NADPH, protochlorophyllide and light. Size-exclusion chromatography on a Superose 6 HR column indicated that MBP-POR has a molecular mass of 155 kDa (compared with the molecular mass of 80 kDa estimated by SDS/PAGE), indicating that it behaves as a dimer in solution. This is the first direct determination of the oligomerization state of POR.
原叶绿素酸酯氧化还原酶（POR）催化原叶绿素酸酯光依赖型还原为叶绿素酸酯，这是叶绿素生物合成途径中的关键反应。为便于进行结构-功能研究，豌豆（Pisum sativum L.）的POR已在大肠杆菌中作为与麦芽糖结合蛋白（MBP）的融合蛋白进行过量表达，其表达量占总可溶性细胞蛋白的5%-10%。通过两步亲和纯化程序，融合蛋白（MBP-POR）已被纯化至均一性大于90%。这代表了首次成功实现植物POR的过量表达和纯化。发现MBP-POR具有活性，并通过连续测定法测定叶绿素酸酯形成速率来确定其动力学性质。Vmax为20.6±0.9 nmol·min⁻¹·mg⁻¹，NADPH和原叶绿素酸酯的Km值分别为8.7±1.9 μM和0.27±0.04 μM。这些结果代表了首次对纯POR的动力学性质进行测定，也是关于双子叶植物POR动力学的首次报道。此处描述的实验表明该酶不是“自杀”酶，催化所需的唯一成分是NADPH、原叶绿素酸酯和光。在Superose 6 HR柱上进行的尺寸排阻色谱表明，MBP-POR的分子量为155 kDa（与SDS/PAGE估计的80 kDa分子量相比），表明它在溶液中表现为二聚体。这是首次直接确定POR的寡聚化状态。
