OBJECTIVETo purify a novel galactose mutarotase (TTE1925) from Thermoanaerobacter tengcongensis for crystallization and X-ray diffraction.

METHODSThe tte 1925 gene was subcloned into the prokaryotic expression vector pGEX-6P-1 and overexpression was obtained in the E.coli BL21 (DE3) through transformation of the right recombinant plasmid that had been verified by colony PCR and sequencing. Soluble fusion protein with glutathione S-transferase tag expressed highly by the induction of isopropyl beta-D-thiogalactoside and was purified in a three-step procedure, which included Glutathione Sepharose 4B affinity, ion chromatography (Resource Q 6 mL), and gel filtration chromatography (10/300 superdex 200).

RESULTThe purity of the purified protein was over 99% and a large amount of claval crystals whose X-ray diffraction reached 1.9 A were obtained.

CONCLUSIONSWe successfully prepared TTE1925 with high purity and obtained crystals for X-ray diffraction. These work paved the way for the further research on the 3-D structure of TTE1925 and its biological mechanism.

Bacterial Proteins ; biosynthesis ; chemistry ; isolation & purification ; Carbohydrate Epimerases ; biosynthesis ; chemistry ; isolation & purification ; Cloning, Molecular ; Crystallization ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Thermoanaerobacter ; enzymology ; genetics ; Transformation, Bacterial

The transition metal cobalt, an essential cofactor for many enzymes in prokaryotes, is taken up by several specific transport systems. The CbiMNQO protein complex belongs to type-1 energy-coupling factor (ECF) transporters and is a widespread group of microbial cobalt transporters. CbiO is the ATPase subunit (A-component) of the cobalt transporting system in the gram-negative thermophilic bacterium Thermoanaerobacter tengcongensis. Here we report the crystal structure of a nucleotide-free CbiO at a resolution of 2.3 Å. CbiO contains an N-terminal canonical nucleotide-binding domain (NBD) and C-terminal helical domain. Structural and biochemical data show that CbiO forms a homodimer mediated by the NBD and the C-terminal domain. Interactions mainly via conserved hydrophobic amino acids between the two C-terminal domains result in formation of a four-helix bundle. Structural comparison with other ECF transporters suggests that non-conserved residues outside the T-component binding groove in the A component likely act as a specificity determinant for T components. Together, our data provide information on understanding of the structural organization and interaction of the CbiMNQO system.
Adenosine Triphosphatases ; chemistry ; Amino Acids ; chemistry ; Biological Transport ; Catalytic Domain ; Cobalt ; chemistry ; Crystallography, X-Ray ; Protein Binding ; Protein Conformation ; Protein Structure, Secondary ; Structure-Activity Relationship ; Thermoanaerobacter ; enzymology