Overexpression, purification, crystallization and preliminary X-ray analysis of putative molybdenum cofactor biosynthesis protein C (MoaC2) from Mycobacterium tuberculosis H37Rv.

Shubhra Srivastava, Vijay Kumar Srivastava, Ashish Arora, J Venkatesh Pratap
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Abstract

Rv0864 (MoaC2) from Mycobacterium tuberculosis is one of the enzymes in the molybdenum cofactor (Moco) biosynthesis pathway. Together with MoaA, MoaC is involved in the conversion of guanosine triphosphate (GTP) to precursor Z, the first step in Moco synthesis. Full-length MoaC2 (17.5 kDa, 167 residues) was cloned in Escherichia coli and purified to homogeneity. Crystals of recombinant M. tuberculosis MoaC2 were grown by vapour diffusion using a hanging-drop setup. Diffracting crystals grew in a condition in which 3 µl protein solution at 10.5 mg ml(-1) was mixed with 1.5 µl reservoir solution (0.025 M potassium sodium tartrate tetrahydrate pH 8.0) and equilibrated against 1000 µl reservoir solution. Diffraction data extending to 2.5 Å resolution were collected at 100 K. The crystal belonged to the cubic space group P2(1)3, with unit-cell parameter 94.5 Å. Matthews coefficient (V(M)) calculations suggested the presence of two molecules in the asymmetric unit, corresponding to a solvent content of about 39%. Molecular-replacement calculations using the E. coli homologue as the search model gave an unambiguous solution.

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结核分枝杆菌 H37Rv 中推测的钼辅助因子生物合成蛋白 C (MoaC2) 的过表达、纯化、结晶和初步 X 射线分析。
结核分枝杆菌中的 Rv0864(MoaC2)是钼辅助因子(Moco)生物合成途径中的酶之一。MoaC 与 MoaA 一起参与将三磷酸鸟苷(GTP)转化为前体 Z,这是 Moco 合成的第一步。在大肠杆菌中克隆了全长的 MoaC2(17.5 kDa,167 个残基),并纯化至均一。重组结核杆菌 MoaC2 的晶体是通过悬滴装置进行蒸汽扩散生长的。衍射晶体生长的条件是 3 µl 蛋白溶液(10.5 mg ml(-1))与 1.5 µl 储液(0.025 M 酒石酸钾钠四水合物 pH 8.0)混合,并与 1000 µl 储液进行平衡。该晶体属于立方空间群 P2(1)3,单位晶胞参数为 94.5 Å。马修斯系数(V(M))计算表明,不对称单元中有两个分子,相当于约 39% 的溶剂含量。以大肠杆菌同源物为搜索模型进行的分子置换计算给出了一个明确的解决方案。
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期刊介绍: Acta Crystallographica Section F is a rapid structural biology communications journal. Articles on any aspect of structural biology, including structures determined using high-throughput methods or from iterative studies such as those used in the pharmaceutical industry, are welcomed by the journal. The journal offers the option of open access, and all communications benefit from unlimited free use of colour illustrations and no page charges. Authors are encouraged to submit multimedia content for publication with their articles. Acta Cryst. F has a dedicated online tool called publBio that is designed to make the preparation and submission of articles easier for authors.
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