Open AccessOverexpression, purification, crystallization and preliminary X‐ray analysis of putative molybdenum cofactor biosynthesis protein C (MoaC2) from Mycobacterium tuberculosis H37Rv
Author(s) -
Srivastava Shubhra,
Srivastava Vijay Kumar,
Arora Ashish,
Pratap J. Venkatesh
Publication year - 2012
Publication title -
acta crystallographica section f
Language(s) - English
Resource type - Journals
ISSN - 1744-3091
DOI - 10.1107/s174430911201665x
Subject(s) - molybdenum cofactor , crystallization , escherichia coli , cofactor , biosynthesis , crystallography , orthorhombic crystal system , molybdenum , chemistry , stereochemistry , biochemistry , biology , nuclear chemistry , enzyme , crystal structure , inorganic chemistry , organic chemistry , gene
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 P 2 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.