During bacteriochlorophyll a biosynthesis, the oxygen-independent conversion of Mg-protoporphyrin IX monomethyl ester (Mg-PME) to protochlorophyllide (Pchlide) is catalyzed by the anaerobic Mg-PME cyclase termed BchE. Bioinformatics analyses in combination with pigment studies of cobalamin-requiring Rhodobacter capsulatus mutants indicated an unusual radical S-adenosylmethionine (SAM) and cobalamin-dependent BchE catalysis. However, in vitro biosynthesis of the isocyclic ring moiety of bacteriochlorophyll using purified recombinant BchE has never been demonstrated. We established a spectroscopic in vitro activity assay which was subsequently validated by HPLC analyses and H218O isotope label transfer onto the carbonyl-group (C-131-oxo) of the isocyclic ring of Pchlide. The reaction product was further converted to chlorophyllide in the presence of light-dependent Pchlide reductase. BchE activity was stimulated by increasing concentrations of NADPH or SAM, and inhibited by S-adenosylhomocysteine. Subcellular fractionation experiments revealed that membrane-localized BchE requires an additional, heat-sensitive cytosolic component for activity. BchE catalysis was not sustained in chimeric experiments when a cytosolic extract from E. coli was used as a substitute. Size-fractionation of the soluble R. capsulatus fraction indicated that enzymatic activity relies on a specific component with an estimated molecular mass between 3 and 10 kDa. A structure guided site-directed mutagenesis approach was performed on the basis of a three-dimensional homology model of BchE. A newly established in vivo complementation assay was used to investigate 24 BchE mutant proteins. Potential ligands of the [4Fe-4S] cluster (Cys204, Cys208, Cys211), of SAM (Phe210, Glu308 and Lys320) and of the proposed cobalamin cofactor (Asp248, Glu249, Leu29, Thr71, Val97) were identified.

中文翻译：

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荚膜红球菌的镁原卟啉IX单甲酯环化酶：细菌SAM的杂环的自由基SAM依赖性合成

在细菌叶绿素a的生物合成过程中，厌氧的Mg-PME环化酶（称为BchE）催化Mg-原卟啉IX单甲酯（Mg-PME）的氧非依赖性转化为原叶绿素（Pchlide）。生物信息学分析与需要钴胺素的荚膜红球菌突变体的色素研究相结合，表明存在异常的自由基S-腺苷甲硫氨酸（SAM）和依赖钴胺素的BchE催化。然而，从未证明使用纯化的重组BchE体外合成细菌叶绿素的杂环部分。我们建立了一种光谱体外活性测定法，该测定法随后通过HPLC分析和将H218O同位素标记转移到Pchlide等价环的羰基（C-131-oxo）上进行了验证。在光依赖性Pchlide还原酶存在下，反应产物进一步转化为叶绿素。通过增加浓度的NADPH或SAM刺激BchE活性，并被S-腺苷同型半胱氨酸抑制。亚细胞分离实验表明，膜定位的BchE需要额外的热敏胞质成分才能发挥活性。当使用大肠杆菌的胞质提取物作为替代品时，在嵌合实验中BchE催化不能持续。可溶性荚膜红球菌级分的大小分级显示酶活性依赖于估计分子量在3至10 kDa之间的特定组分。基于BchE的三维同源性模型，进行了结构指导的定点诱变方法。新建立的体内互补测定法用于研究24种BchE突变蛋白。确定了[4Fe-4S]簇（Cys204，Cys208，Cys211），SAM（Phe210，Glu308和Lys320）和拟议的钴胺素辅因子（Asp248，Glu249，Leu29，Thr71，Val97）的潜在配体。