A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis -: Reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus
A second nitrogenase-like enzyme for bacteriochlorophyll biosynthesis -: Reconstitution of chlorophyllide a reductase with purified X-protein (BchX) and YZ-protein (BchY-BchZ) from Rhodobacter capsulatus
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DOI：
10.1074/jbc.m601750200
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发表时间：
2006-05-26
影响因子：
4.8
通讯作者：
Fujita, Yuichi
中科院分区：
文献类型：
--
作者：
Nomata, Jiro;Mizoguchi, Tadashi;Fujita, Yuichi
在大多数光合生物中，叶绿素a的二氢卟酚环结构是通过暗操作固氮酶样酶原叶绿素还原酶（DPOR）还原卟啉D环形成的。随后，二氢卟酚B环在细菌叶绿素生物合成中被还原以形成细菌二氢卟酚环结构。缺乏三个基因，bchX，bchY，或bchZ，这表明显着的序列相似性的固氮酶的结构基因之一的突变体的表型分析表明，第二个固氮酶样酶参与的二氢卟酚B环还原。然而，没有生物化学证据证明这一点。在这里，我们报告的重构叶绿素a还原酶（COR）与纯化的蛋白质。分离出两种过表达Strep标记的BchX和BchY的荚膜红杆菌菌株。将Strep标记的BchX纯化为单一多肽，并将BchZ与Strep标记的BchY共纯化。当BchX和BchY-BchZ组分与叶绿素a、ATP和连二亚硫酸盐在厌氧条件下孵育时，叶绿素a在80%丙酮中转化为具有在734 nm（P734）处的较长波长的Qy带的新色素。P734的形成依赖于ATP和连二亚硫酸盐。高效液相色谱和质谱分析表明，P734是3-乙烯基菌叶绿酸a，它是由叶绿素a的B环还原形成的。这些结果表明，二氢卟酚的B环被第二种固氮酶样酶还原，并且两种固氮酶样酶DPOR和COR的顺序作用将卟啉转化为菌绿素。固氮酶样酶的进化意义，以确定（细菌）叶绿素色素的环结构进行了讨论。
In most photosynthetic organisms, the chlorin ring structure of chlorophyll a is formed by the reduction of the porphyrin D-ring by the dark-operative nitrogenase-like enzyme, protochlorophyllide reductase ( DPOR). Subsequently, the chlorin B-ring is reduced in bacteriochlorophyll biosynthesis to form a bacteriochlorin ring structure. Phenotypic analysis of mutants lacking one of three genes, bchX, bchY, or bchZ, which show significant sequence similarity to the structural genes of nitrogenase, suggests that a second nitrogenase-like enzyme is involved in the chlorin B-ring reduction. However, there is no biochemical evidence for this. Here, we report the reconstitution of chlorophyllide a reductase ( COR) with purified proteins. Two Rhodobacter capsulatus strains that overexpressed Strep-tagged BchX and BchY were isolated. Strep-tagged BchX was purified as a single polypeptide, and BchZ was co-purified with Strep-tagged BchY. When BchX and BchY-BchZ components were incubated with chlorophyllide a, ATP, and dithionite under anaerobic conditions, chlorophyllide a was converted to a new pigment with a Qy band of longer wavelength at 734 nm ( P734) in 80% acetone. The formation of P734 was dependent on ATP and dithionite. High performance liquid chromatography and mass spectroscopic analysis indicated that P734 is 3-vinyl bacteriochlorophyllide a, which is formed by the B-ring reduction of chlorophyllide a. These results demonstrate that the B-ring of chlorin is reduced by a second nitrogenase-like enzyme and that the sequential actions of two nitrogenase-like enzymes, DPOR and COR, convert porphyrin to bacteriochlorin. The evolutionary implications of nitrogenase-like enzymes to determine the ring structure of ( bacterio) chlorophyll pigments are discussed.