Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus
Substrate Recognition of Nitrogenase-like Dark Operative Protochlorophyllide Oxidoreductase from Prochlorococcus marinus
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DOI：
10.1074/jbc.m805206200
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发表时间：
2008-10-31
影响因子：
4.8
通讯作者：
Jahn, Dieter
中科院分区：
文献类型：
--
作者：
Broecker, Markus J.;Waetzlich, Denise;Jahn, Dieter
叶绿素和细菌叶绿素生物合成需要原叶绿素内酯氧化还原酶对原叶绿素a环D进行双电子还原，形成叶绿素a。已知光依赖性(光依赖性Pchlide氧化还原酶(LPOR))和不相关的暗操作酶(暗操作Pchlide氧化还原酶(DPOR))。在无光条件下，DPOR 在裸子植物、苔藓、蕨类植物、藻类和光合细菌的叶绿素生物合成中发挥着重要作用。尽管 DPOR 与固氮酶具有显着的氨基酸序列同源性，但只有最初的催化步骤类似于固氮酶催化。底物配位和随后的[Fe-S]簇依赖性催化被认为是不相关的。在这里，我们表征了第一个蓝藻 DPOR，其由同二聚体蛋白质复合物 ChlL(2) 和异四聚体蛋白质复合物 (ChlNB)(2) 组成。 ChlL2 二聚体包含一个 EPR 活性 [4Fe-4S] 簇，而 (ChlNB)2 复合物表现出两个 [4Fe-4S] 簇的 EPR 信号，其 g 值和温度依赖性弛豫行为存在差异。这些发现表明 (ChlNB) 2 中发现的各个 [4Fe-4S] 簇的几何形状存在变化。为了分析 DPOR 底物识别，测试了 11 种在四个吡咯环和等环环上具有改变取代基的合成衍生物以及八种叶绿素生物合成中间体作为 DPOR 底物。尽管 DPOR 可以容忍环 A-C 上环取代基的微小修饰，但显然发现催化目标环 D 具有高特异性配位。此外，原叶绿素a、相应的[8-乙烯基]-衍生物和原叶绿素b同样用作底物。观察到与 LPOR 结合底物的明显差异。推导了蓝藻叶绿素生物合成的替代生物合成途径，涉及 C8-乙烯基的还原和叶绿素 a/b 型 C7 甲基/甲酰基的相互转化。
Chlorophyll and bacteriochlorophyll biosynthesis requires the two-electron reduction of protochlorophyllide a ring D by a protochlorophyllide oxidoreductase to form chlorophyllide a. A light-dependent (light-dependent Pchlide oxidoreductase (LPOR)) and an unrelated dark operative enzyme (dark operative Pchlide oxidoreductase (DPOR)) are known. DPOR plays an important role in chlorophyll biosynthesis of gymnosperms, mosses, ferns, algae, and photosynthetic bacteria in the absence of light. Although DPOR shares significant amino acid sequence homologies with nitrogenase, only the initial catalytic steps resemble nitrogenase catalysis. Substrate coordination and subsequent [Fe-S] cluster-dependent catalysis were proposed to be unrelated. Here we characterized the first cyanobacterial DPOR consisting of the homodimeric protein complex ChlL(2) and a heterotetrameric protein complex (ChlNB)(2). The ChlL2 dimer contains one EPR active [4Fe-4S] cluster, whereas the (ChlNB) 2 complex exhibited EPR signals for two [ 4Fe-4S] clusters with differences in their g values and temperature-dependent relaxation behavior. These findings indicate variations in the geometry of the individual [4Fe-4S] clusters found in (ChlNB) 2. For the analysis of DPOR substrate recognition, 11 synthetic derivatives with altered substituents on the four pyrrole rings and the isocyclic ring plus eight chlorophyll biosynthetic intermediates were tested as DPOR substrates. Although DPOR tolerated minor modifications of the ring substituents on rings A-C, the catalytic target ring D was apparently found to be coordinated with high specificity. Furthermore, protochlorophyllide a, the corresponding [8-vinyl]-derivative and protochlorophyllide b were equally utilized as substrates. Distinct differences from substrate binding by LPOR were observed. Alternative biosynthetic routes for cyanobacterial chlorophyll biosynthesis with regard to the reduction of the C8-vinyl group and the interconversion of a chlorophyll a/b type C7 methyl/formyl group were deduced.