The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis
The role of protein surface charge in catalytic activity and chloroplast membrane association of the pea NADPH: protochlorophyllide oxidoreductase (POR) as revealed by alanine scanning mutagenesis
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丙氨酸扫描诱变揭示了蛋白质表面电荷在豌豆 NADPH 催化活性和叶绿体膜缔合中的作用：原叶绿素氧化还原酶 (POR)
DOI：
10.1023/a:1006135100760
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发表时间：
2004
影响因子：
5.1
通讯作者：
M. Timko
中科院分区：
文献类型：
--
作者：
C. Dahlin;H. Aronsson;H. Wilks;N. Lebedev;C. Sundqvist;M. Timko
NADPH：原叶绿素氧化还原酶（POR）催化叶绿素生物合成中原叶绿素（pchlide）到叶绿素（chlide）的光依赖性还原。POR是一种外周膜蛋白，其在维管植物质体的原片层体中积累到高水平，并且在发育和成熟质体的类囊体膜中以低水平存在。豌豆（Pisum sativum L.）进行POR，并分析所得的突变体酶在体内催化pchlide光转化的能力，并在体外与类囊体膜制剂适当地关联。在37种突变酶中，5种保留了野生型活性水平，14种无催化活性，其余18种表现出功能水平的改变。几种突变酶显示出温度依赖性酶活性，在32 °C下无活性，但在24 °C下部分有活性。预测的蛋白质α-螺旋区的突变对酶活性的影响最小，而预测的蛋白质β-折叠区的突变对酶功能的不利影响一致。在没有添加NADPH的情况下，无论是野生型POR还是任何突变体POR都不能抵抗嗜热菌蛋白酶在类囊体膜上组装后的蛋白水解。相比之下，当NADPH存在于测定混合物中时，发现检查的37个突变体POR中的13个在处理后对嗜热菌蛋白酶具有抗性，这表明突变不影响它们正确附着于类囊体膜的能力。在一般情况下，在最N-和C-末端区域的成熟蛋白质的丙氨酸取代带电的氨基酸没有显着影响POR组件，而在蛋白质的中心核心（残基86和342之间）的突变是不能正确的附件的类囊体。未能以蛋白酶抗性方式与类囊体膜正确结合与催化功能的丧失仅弱相关。这些研究是确定POR功能和细胞器内定位的关键结构决定因素的第一步。
NADPH:protochlorophyllide oxidoreductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (pchlide) to chlorophyllide (chlide) in the biosynthesis of chlorophyll. POR is a peripheral membrane protein that accumulates to high levels in the prolamellar bodies of vascular plant etioplasts and is present at low levels in the thylakoid membranes of developing and mature plastids. Clustered charged-to-alanine scanning mutagenesis of the pea (Pisum sativum L.) POR was carried out and the resulting mutant enzymes analyzed for their ability to catalyze pchlide photoconversion in vivo and to associate properly with thylakoid membrane preparations in vitro. Of 37 mutant enzymes examined, 5 retained wild-type levels of activity, 14 were catalytically inactive, and the remaining 18 exhibited altered levels of function. Several of the mutant enzymes showed temperature- dependent enzymatic activity, being inactive at 32 °C, but partially active at 24 °C. Mutations in predicted α- helical regions of the protein showed the least effect on enzyme activity, whereas mutations in predicted β-sheet regions of the protein showed a consistent adverse affect on enzyme function. In the absence of added NADPH, neither wild-type POR nor any of the mutant PORs resisted proteolysis by thermolysin following assembly onto the thylakoid membranes. In contrast, when NADPH was present in the assay mixture, 13 of the 37 mutant PORs examined were found to be resistant to thermolysin upon treatment, suggesting that the mutations did not affect their ability to be properly attached to the thylakoid membrane. In general, the replacement of charged amino acids by alanine in the most N- and C-terminal regions of the mature protein did not significantly affect POR assembly, whereas mutations within the central core of the protein (between residues 86 and 342) were incapable of proper attachment to the thylakoid. Failure to properly associate with the thylakoid membrane in a protease resistant manner was only weakly correlated to loss of catalytic function. These studies are a first step towards defining structural determinants crucial to POR function and intraorganellar localization.
影响因子：
4.1
作者：
CHENEVERT, SW;FOSSETT, NG;LEE, WR
通讯作者：
LEE, WR
影响因子：
2.9
作者：
JORNVALL, H;PERSSON, B;GHOSH, D
通讯作者：
GHOSH, D
蛋白质中的羧乙基赖氨酸：天然羰基还原酶/NADP( )依赖性前列腺素脱氢酶。
DOI：
10.1073/pnas.90.2.502
发表时间：
1993
影响因子：
11.1
作者：
Krook,M;Ghosh,D;Strömberg,R;Carlquist,M;Jörnvall,H
通讯作者：
Jörnvall,H