ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis
ATP-driven reduction by dark-operative protochlorophyllide oxidoreductase from Chlorobium tepidum mechanistically resembles nitrogenase catalysis
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DOI：
10.1074/jbc.m708010200
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发表时间：
2008-04-18
影响因子：
4.8
通讯作者：
Moser, Juergen
中科院分区：
文献类型：
--
作者：
Broecker, Markus J.;Virus, Simone;Moser, Juergen
在裸子植物、藻类和光合细菌的叶绿素和细菌叶绿素生物合成过程中，暗操作原叶绿素内酯氧化还原酶 (DPOR) 立体定向地还原芳香族原叶绿素内酯的 D 环以产生叶绿素。我们描述了大肠杆菌中温绿菌中 DPOR 亚基 BchN、BchB 和 BchL 的异源过量生产，从而使它们的纯化达到明显的同质性。催化活性为3.15 nmol min(-1)mg(-1)，原叶绿素的K-m值为6.1 μM，ATP为13.5 μM，还原剂连二亚硫酸盐的K-m值为52.7 μM。为了鉴定对 DPOR 功能重要的残基，通过定点诱变生成了 21 种酶变体，并研究了它们的金属含量、光谱特征和催化活性。发现同型二聚体 BchL(2) 的两个半胱氨酸残基（Cys(97) 和 Cys(131)）协调亚基间 [4Fe-4S] 簇，对于低电位电子转移至 (BchNB)(2) 作为原叶绿素底物还原的一部分至关重要。同样，Lys(10) 和 Leu(126) 对于 ATP 驱动的 BchL(2) 电子转移至关重要。 DPOR电子转移的活化能为22.2 kJ mol(-1)，表明每个催化循环需要4个ATP。在氨基酸水平上，BchL 与固氮酶亚基 NifH 具有 33% 的相同性，从而可以提出第一个暂定结构模型。在 (BchNB)(2) 中，我们发现四个半胱氨酸残基，其中三个来自 BchN（Cys(21)、Cys(46) 和 Cys(103)），一个来自 BchB（Cys(94)），协调催化所需的第二个亚基间 [4Fe-4S] 簇。没有发现任何类型的含钼辅因子的证据，表明 DPOR 亚基 BchN 明显不同于同源固氮酶亚基 NifD。根据现有数据，我们提出了 DPOR 的酶促机制。
During chlorophyll and bacteriochlorophyll biosynthesis in gymnosperms, algae, and photosynthetic bacteria, dark-operative protochlorophyllide oxidoreductase (DPOR) reduces ring D of aromatic protochlorophyllide stereospecifically to produce chlorophyllide. We describe the heterologous overproduction of DPOR subunits BchN, BchB, and BchL from Chlorobium tepidum in Escherichia coli allowing their purification to apparent homogeneity. The catalytic activity was found to be 3.15 nmol min(-1)mg(-1) with K-m values of 6.1 mu M for protochlorophyllide, 13.5 mu M for ATP, and 52.7 mu M for the reductant dithionite. To identify residues important in DPOR function, 21 enzyme variants were generated by site-directed mutagenesis and investigated for their metal content, spectroscopic features, and catalytic activity. Two cysteine residues (Cys(97) and Cys(131)) of homodimeric BchL(2) are found to coordinate an intersubunit [4Fe-4S] cluster, essential for low potential electron transfer to (BchNB)(2) as part of the reduction of the protochlorophyllide substrate. Similarly, Lys(10) and Leu(126) are crucial to ATP-driven electron transfer from BchL(2). The activation energy of DPOR electron transfer is 22.2 kJ mol(-1) indicating a requirement for 4 ATP per catalytic cycle. At the amino acid level, BchL is 33% identical to the nitrogenase subunit NifH allowing a first tentative structural model to be proposed. In (BchNB)(2), we find that four cysteine residues, three from BchN (Cys(21), Cys(46), and Cys(103)) and one from BchB (Cys(94)), coordinate a second intersubunit [4Fe-4S] cluster required for catalysis. No evidence for any type of molybdenum-containing cofactor was found, indicating that the DPOR subunit BchN clearly differs from the homologous nitrogenase subunit NifD. Based on the available data we propose an enzymatic mechanism of DPOR.