Watanabe N, Che F S, Iwano M, Takayama S, Yoshida S, Isogai A
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan.
J Biol Chem. 2001 Jun 8;276(23):20474-81. doi: 10.1074/jbc.M101140200. Epub 2001 Mar 23.
Protoporphyrinogen oxidase (Protox) is the final enzyme in the common pathway of chlorophyll and heme biosynthesis. Two Protox isoenzymes have been described in tobacco, a plastidic and a mitochondrial form. We isolated and sequenced spinach Protox cDNA, which encodes a homolog of tobacco mitochondrial Protox (Protox II). Alignment of the deduced amino acid sequence between Protox II and other tobacco mitochondrial Protox homologs revealed a 26-amino acid N-terminal extension unique to the spinach enzyme. Immunoblot analysis of spinach leaf extract detected two proteins with apparent molecular masses of 57 and 55 kDa in chloroplasts and mitochondria, respectively. In vitro translation experiments indicated that two translation products (59 and 55 kDa) are produced from Protox II mRNA, using two in-frame initiation codons. Transport experiments using green fluorescent protein-fused Protox II suggested that the larger and smaller translation products (Protox IIL and IIS) target exclusively to chloroplasts and mitochondria, respectively.
原卟啉原氧化酶（Protox）是叶绿素和血红素生物合成共同途径中的最后一种酶。烟草中已描述了两种Protox同工酶，一种是质体形式，另一种是线粒体形式。我们分离并测序了菠菜Protox cDNA，其编码烟草线粒体Protox（Protox II）的同源物。Protox II与其他烟草线粒体Protox同源物之间推导的氨基酸序列比对显示，菠菜酶具有一个26个氨基酸的N端延伸，这是其特有的。对菠菜叶提取物的免疫印迹分析分别在叶绿体和线粒体中检测到两种表观分子量为57 kDa和55 kDa的蛋白质。体外翻译实验表明，使用两个读框内起始密码子，Protox II mRNA产生两种翻译产物（59 kDa和55 kDa）。使用绿色荧光蛋白融合的Protox II进行的转运实验表明，较大和较小的翻译产物（Protox IIL和IIS）分别仅靶向叶绿体和线粒体。
