Krengel Ute, Dey Raja, Sasso Severin, Okvist Mats, Ramakrishnan Chandra, Kast Peter
Department of Chemistry and Bioscience, Chalmers University of Technology, PO Box 462, SE-40530 Göteborg, Sweden.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2006 May 1;62(Pt 5):441-5. doi: 10.1107/S1744309106012036. Epub 2006 Apr 12.
Chorismate mutase catalyzes the conversion of chorismate to prephenate in the biosynthesis of the aromatic amino acids tyrosine and phenylalanine in bacteria, fungi and plants. Here, the crystallization of the unusual secreted chorismate mutase from Mycobacterium tuberculosis (encoded by Rv1885c), a 37.2 kDa dimeric protein belonging to the AroQ(gamma) subclass of mutases, is reported. Crystal optimization was non-trivial and is discussed in detail. To obtain crystals of sufficient quality, it was critical to initiate crystallization at higher precipitant concentration and then transfer the drops to lower precipitant concentrations within 5-15 min, in an adaptation of a previously described technique [Saridakis & Chayen (2000), Protein Sci. 9, 755-757]. As a result of the optimization, diffraction improved from 3.5 to 1.3 A resolution. The crystals belong to space group P2(1), with unit-cell parameters a = 42.6, b = 72.6, c = 62.0 angstroms, beta = 104.5 degrees. The asymmetric unit contains one biological dimer, with 167 amino acids per protomer. A soak with a transition-state analogue is also described.
分支酸变位酶在细菌、真菌和植物中芳香族氨基酸酪氨酸和苯丙氨酸的生物合成过程中催化分支酸转化为预苯酸。本文报道了结核分枝杆菌中一种不同寻常的分泌型分支酸变位酶（由Rv1885c编码）的晶体结构，该酶是一种37.2 kDa的二聚体蛋白，属于变位酶的AroQ(γ)亚类。晶体优化过程并非易事，将对此进行详细讨论。为了获得足够质量的晶体，关键是在较高沉淀剂浓度下开始结晶，然后在5 - 15分钟内将液滴转移到较低沉淀剂浓度下，这是对先前描述技术的一种改进[Saridakis & Chayen (2000), Protein Sci. 9, 755 - 757]。经过优化，衍射分辨率从3.5 Å提高到了1.3 Å。晶体属于空间群P2(1)，晶胞参数为a = 42.6、b = 72.6、c = 62.0 Å，β = 104.5°。不对称单元包含一个生物学二聚体，每个原体有167个氨基酸。本文还描述了用过渡态类似物进行浸泡的情况。
