Hädener A, Matzinger P K, Malashkevich V N, Louie G V, Wood S P, Oliver P, Alefounder P R, Pitt A R, Abell C, Battersby A R
Institut für Organische Chemie, Universität, Basel, Switzerland.
Eur J Biochem. 1993 Feb 1;211(3):615-24. doi: 10.1111/j.1432-1033.1993.tb17589.x.
Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocycles. In the course of an investigation of the crystal structure of this enzyme, we intended to follow a new strategy to obtain the X-ray phase information, i.e. the collection of multiwavelength anomalous diffraction data from a crystal of a seleno-L-methionine (SeMet)-labelled variant of the protein. We have expressed and purified HMBS from Escherichia coli (34268 Da) in which all (six) methionine (Met) residues are replaced by SeMet. Complete replacement, as shown by amino acid composition analysis and by electrospray mass spectrometry, was achieved by growing the Met-requiring mutant E. coli PO1562 carrying the plasmid pPA410 in a medium containing 50 mg/l SeMet as the sole source of Met. [SeMet]HMBS exhibits full enzyme activity, as reflected by unchanged steady-state kinetic parameters relative to native enzyme. Rhombohedral crystals of [SeMet]HMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mM EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bristris-propane buffer were equilibrated by vapour diffusion at 20 degrees C against reservoirs of saturated NaCl). However, being very thin plates, these crystals were not suitable for X-ray analysis. Alternatively, rectangular crystals were obtained at pH 5.3 using conditions based on those reported for wild-type HMBS [sitting drops of 50 microliters containing 6-7 mg/ml protein, 0.3 mM EDTA, 15 mM dithiothreitol, 10% (mass/vol.) poly(ethylene glycol) 6000 and 0.01% NaN3 in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20 degrees C against a reservoir of 10-20 mg solid dithiothreitol]. X-ray diffraction data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that [SeMet]HMBS and native HMBS crystallise isomorphously. A difference Fourier map using FSeMet-Fnative and phases derived from the native structure, which has recently been determined independently by multiple isomorphous replacement, showed positive difference peaks centered at or close to where the sulphur atoms of the Met side chains appear in the native structure. In addition, paired positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational differences in the active site, probably due to differences in the state of oxidation of the cofactor in the two crystalline samples.
羟甲基胆色素原合酶（HMBS）催化胆色素原转化为羟甲基胆色素，后者是血红素、叶绿素、维生素B12及相关大环化合物生物合成过程中的一种线性四吡咯中间体。在对该酶晶体结构的研究过程中，我们打算采用一种新策略来获取X射线相位信息，即从硒代-L-甲硫氨酸（SeMet）标记的蛋白质变体晶体中收集多波长反常衍射数据。我们已从大肠杆菌（34268 Da）中表达并纯化了HMBS，其中所有（六个）甲硫氨酸（Met）残基均被SeMet取代。通过在含有50 mg/l SeMet作为唯一甲硫氨酸来源的培养基中培养携带质粒pPA410的甲硫氨酸需求型突变大肠杆菌PO1562，经氨基酸组成分析和电喷雾质谱分析表明实现了完全取代。[SeMet]HMBS表现出完全的酶活性，相对于天然酶，稳态动力学参数未发生变化即反映了这一点。[SeMet]HMBS的菱形晶体可在该酶的最适pH（7.4）下生长（含有30 mg/ml蛋白质、0.4 mM EDTA、20 mM二硫苏糖醇、3 M NaCl和15 mM Bristris-丙烷缓冲液的溶液在20℃下通过气相扩散与饱和NaCl储液平衡）。然而，这些晶体是非常薄的平板，不适合进行X射线分析。另外，在pH 5.3条件下，基于报道的野生型HMBS的条件获得了矩形晶体[含有6 - 7 mg/ml蛋白质、0.3 mM EDTA、15 mM二硫苏糖醇、10%（质量/体积）聚乙二醇6000和0.01%叠氮化钠的50微升坐滴在0.1 M醋酸钠中，于20℃下通过气相扩散与10 - 20 mg固体二硫苏糖醇储液平衡]。晶体的X射线衍射数据在0.21 nm分辨率下完成度达到93.8%，表明[SeMet]HMBS和天然HMBS同晶型结晶。使用FSeMet - F天然以及源自天然结构的相位（最近已通过多重同晶置换独立确定）得到的差分傅里叶图显示，正差分峰集中在天然结构中甲硫氨酸侧链硫原子出现的位置或其附近。此外，在HMBS辅因子附近的差分图中的成对正/负峰表明活性位点存在构象差异，这可能是由于两个结晶样品中辅因子氧化态不同所致。
