Crystal structure of the L protein of Rhodobacter sphaeroides light-independent protochlorophyllide reductase with MgADP bound: a homologue of the nitrogenase Fe protein.
Crystal structure of the L protein of Rhodobacter sphaeroides light-independent protochlorophyllide reductase with MgADP bound: a homologue of the nitrogenase Fe protein.
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DOI：
10.1021/bi801058r
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发表时间：
2008-12
期刊：
影响因子：
2.9
通讯作者：
Ranjana Sarma;B. M. Barney;T. Hamilton;Alma Jones;L. Seefeldt;J. W. Peters
中科院分区：
文献类型：
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作者：
Ranjana Sarma;B. M. Barney;T. Hamilton;Alma Jones;L. Seefeldt;J. W. Peters
球形红细菌暗反应原叶绿素酸酯还原酶（DPOR）的L蛋白（BchL）已从棕色固氮菌表达系统中纯化出来；其与核苷酸的相互作用已被检测，并且该蛋白结合MgADP的X射线结构已被解析到1.6 Å分辨率。DPOR催化原叶绿素酸酯还原为叶绿素酸酯，这是细菌叶绿素生物合成的关键反应。DPOR全酶由两种组分蛋白组成，即二聚体BchL蛋白和异源四聚体BchN/BchB蛋白。DPOR组分蛋白与固氮酶铁蛋白（NifH）和钼铁（NifDK）蛋白在整体上有显著的相似性，固氮酶是负责将氮气还原为氨的酶系统。在此，BchL在棕色固氮菌中表达，并使用工程化的多聚组氨酸标签纯化至均一。纯化的重组BchL被发现每摩尔BchL同二聚体含有3.6摩尔铁，这与一个[4Fe - 4S]簇的存在相符，并且类似于铁蛋白中存在的[4Fe - 4S]簇。通过铁螯合测定检测了MgATP和MgADP诱导的BchL构象变化，发现其与铁蛋白中观察到的核苷酸刺激的铁释放明显不同。重组BchL与结合的MgADP一起结晶，其结构被解析到1.6 Å分辨率。发现BchL与固氮酶铁蛋白在整体结构上具有相似性，包括亚基桥连的[4Fe - 4S]簇和核苷酸结合位点。然而，尽管结构相似性很高，但在固氮酶底物还原测定中发现BchL不能替代铁蛋白。新解析的BchL结构及其与它的近亲固氮酶铁蛋白的比较，为理解这些高度相关的蛋白如何在微生物系统中区分它们各自的功能提供了基础，在微生物系统中每种蛋白都必须同时发挥作用。
The L protein (BchL) of the dark-operative protochlorophyllide reductase (DPOR) from Rhodobacter sphaeroides has been purified from an Azotobacter vinelandii expression system; its interaction with nucleotides has been examined, and the X-ray structure of the protein has been determined with bound MgADP to 1.6 A resolution. DPOR catalyzes the reduction of protochlorophyllide to chlorophyllide, a reaction critical to the biosynthesis of bacteriochlorophylls. The DPOR holoenzyme is comprised of two component proteins, the dimeric BchL protein and the heterotetrameric BchN/BchB protein. The DPOR component proteins share significant overall similarities with the nitrogenase Fe protein (NifH) and the MoFe (NifDK) protein, the enzyme system responsible for reduction of dinitrogen to ammonia. Here, BchL was expressed in A. vinelandii and purified to homogeneity using an engineered polyhistidine tag. The purified, recombinant BchL was found to contain 3.6 mol of Fe/mol of BchL homodimer, consistent with the presence of a [4Fe-4S] cluster and analogous to the [4Fe-4S] cluster present in the Fe protein. The MgATP- and MgADP-induced conformational changes in BchL were examined by an Fe chelation assay and found to be distinctly different from the nucleotide-stimulated Fe release observed for the Fe protein. The recombinant BchL was crystallized with bound MgADP, and the structure was determined to 1.6 A resolution. BchL is found to share overall structural similarity with the nitrogenase Fe protein, including the subunit bridging [4Fe-4S] cluster and nucleotide binding sites. Despite the high level of structural similarity, however, BchL is found to be incapable of substituting for the Fe protein in a nitrogenase substrate reduction assay. The newly determined structure of BchL and its comparison to its close homologue, the nitrogenase Fe protein, provide the basis for understanding how these highly related proteins can discriminate between their respective functions in microbial systems where each must function simultaneously.