School of Bioagricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Plant Cell Physiol. 2011 Nov;52(11):1983-93. doi: 10.1093/pcp/pcr132. Epub 2011 Sep 24.
Dark-operative protochlorophyllide (Pchlide) oxidoreductase (DPOR) is a nitrogenase-like enzyme consisting of the two components, L-protein (a ChlL dimer) and NB-protein (a ChlN-ChlB heterotetramer), to catalyze Pchlide reduction in Chl biosynthesis. While nitrogenase is distributed only among certain prokaryotes, the probable structural genes for DPOR are encoded by chloroplast DNA in lower plants. Here we show functional evaluation of DPOR encoded by chloroplast DNA in a moss Physcomitrella patens by the complementation analysis of the cyanobacterium Leptolyngbya boryana and the heterologous reconstitution of the moss L-protein and the cyanobacterial NB-protein. Two shuttle vectors to overexpress chlL and chlN-chlB from P. patens were introduced into the cyanobacterial chlL- and chlB-lacking mutants, respectively. Both transformants restored the ability to perform Chl biosynthesis in the dark, indicating that the chloroplast-encoded DPOR components form an active complex with the cyanobacterial components. The L-protein of P. patens was purified from the cyanobacterial transformant, and DPOR activity was reconstituted in a heterologous combination with the cyanobacterial NB-protein. The specific activity of the L-protein from P. patens was determined to be 118 nmol min(-1) mg (-1), which is even higher than that of the cyanobacterial L-protein (76 nmol min(-1) mg (-1)). Upon exposure to air, the activity of the L-protein from P. patens decayed with a half-life of 30 s, which was eight times faster than that of the cyanobacterial L-protein (240 s). These results suggested that the chloroplast-encoded L-protein functions as efficiently as the cyanobacterial L-protein but is more oxygen labile than the cyanobacterial L-protein.
暗反应原叶绿素酸酯（Pchlide）氧化还原酶（DPOR）是一种类似于氮酶的酶，由两个组件组成，L-蛋白（一个 ChlL 二聚体）和 NB-蛋白（一个 ChlN-ChlB 杂四聚体），以催化叶绿素生物合成中的 Pchlide 还原。虽然氮酶仅分布在某些原核生物中，但 DPOR 的可能结构基因是由低等植物的叶绿体 DNA 编码的。在这里，我们通过蓝藻 Leptolyngbya boryana 的互补分析和苔藓 L-蛋白和蓝藻 NB-蛋白的异源重组，对苔藓 Physcomitrella patens 中的叶绿体 DNA 编码的 DPOR 进行了功能评估。两个穿梭载体分别用于在蓝藻 chlL 和 chlB 缺失突变体中过表达来自 P. patens 的 chlL 和 chlN-chlB。这两个转化体都恢复了在黑暗中进行叶绿素生物合成的能力，表明叶绿体编码的 DPOR 组件与蓝藻组件形成了一个活性复合物。从蓝藻转化体中纯化了来自 P. patens 的 L-蛋白，并与蓝藻的 NB-蛋白进行了异源重组，恢复了 DPOR 活性。从 P. patens 纯化的 L-蛋白的比活性被确定为 118 nmol min(-1) mg(-1)，甚至高于蓝藻的 L-蛋白（76 nmol min(-1) mg(-1)）。暴露在空气中时，P. patens 的 L-蛋白的活性会以半衰期 30 秒的速度衰减，这比蓝藻的 L-蛋白（240 秒）快八倍。这些结果表明，叶绿体编码的 L-蛋白的功能与蓝藻的 L-蛋白一样有效，但比蓝藻的 L-蛋白更容易被氧气破坏。
