Expression, crystallization and preliminary crystallographic data analysis of PigF, an O-methyltransferase from the prodigiosin-synthetic pathway in Serratia
Expression, crystallization and preliminary crystallographic data analysis of PigF, an O-methyltransferase from the prodigiosin-synthetic pathway in Serratia
复制标题
PigF（一种来自沙雷氏菌灵菌红合成途径的 O-甲基转移酶）的表达、结晶和初步晶体学数据分析
DOI：
10.1107/s1744309112024001
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发表时间：
2012-08-01
影响因子：
0.9
通讯作者：
Xu, Dongqing
中科院分区：
文献类型：
--
作者：
Liu, Shaowei;Ran, Tingting;Xu, Dongqing
Prodigiosin 是 prodiginines 的一员，是一种红色线性三吡咯化合物。已在沙雷氏菌中鉴定出用于生物合成灵菌红的基因簇，并且该簇中的大多数基因已被功能指定。此前已经确定了灵菌红素的分叉生物合成途径。 4-甲氧基-2,2'-联吡咯-5-甲醛(MBC)生物合成途径的最后一步是由PigF催化，将甲基转移到4-羟基-2,2'-联吡咯-5-甲醛(HBC)上形成最终产物MBC，但其催化机制尚不清楚。为了阐明其机制，对重组 PigF 进行了纯化和结晶。该晶体属于P21空间群，晶胞参数a=69.4，b=52.4，c相似=相似279.2埃，β=96.8度。天然晶体可能含有六个不对称单元分子，VM为2.17埃3Da-1，溶剂含量为43.43%。使用中华人民共和国上海同步辐射装置 (SSRF) 的光束线 BL17U 上的同步辐射，以 2.6 埃分辨率收集了完整的数据集。分子替代不成功。为了通过实验定相解析PigF的结构，在0.01 M亚精胺作为添加剂的条件下制备了硒代蛋氨酸衍生的蛋白质晶体。一颗晶体衍射至 1.9 埃分辨率，并在 SSRF 的光束线 BL17U 上收集了完整的数据集。该晶体属于P21空间群，晶胞参数a=69.0，b=52.9，c=93.4埃，β近似=近似97.3度。重原子亚结构测定和SAD定相清楚地表明该晶体在不对称单元中含有两个分子，VM为2.19埃3 Da-1，溶剂含量为43.82%。
Prodigiosin, which is a member of the prodiginines, is a red linear tripyrrole compound. A gene cluster for the biosynthesis of prodigiosin has been identified in Serratia and most genes in the cluster have been functionally assigned. A bifurcated biosynthetic pathway for prodigiosin has previously been determined. The last step in the biosynthetic pathway of 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) is catalyzed by PigF, which transfers a methyl group to 4-hydroxy-2,2'-bipyrrole-5-carbaldehyde (HBC) to form the terminal product MBC, but its catalytic mechanism is not known. To elucidate its mechanism, recombinant PigF was purified and crystallized. The crystals belonged to space group P21, with unit-cell parameters a = 69.4, b = 52.4, c similar to=similar to 279.2 angstrom, beta=96.8 degrees. The native crystals may contain six molecules in the asymmetric unit, with a VM of 2.17 angstrom 3 Da-1 and a solvent content of 43.43%. A full data set was collected at 2.6 angstrom resolution using synchrotron radiation on beamline BL17U of Shanghai Synchrotron Radiation Facility (SSRF), People's Republic of China. Molecular replacement was unsuccessful. To solve the structure of PigF by experimental phasing, selenomethionine-derivativized protein crystals were prepared from a condition with 0.01 M spermidine as an additive. One crystal diffracted to 1.9 angstrom resolution and a full data set was collected on beamline BL17U at SSRF. The crystal belonged to space group P21, with unit-cell parameters a = 69.0, b = 52.9, c = 93.4 angstrom, beta similar to=similar to 97.3 degrees. Heavy-atom substructure determination and phasing by SAD clearly showed that the crystal contains two molecules in the asymmetric unit, with a VM of 2.19 angstrom 3 Da-1 and a solvent content of 43.82%.