Purification, crystallization and preliminary X-ray diffraction analysis of the catalytic domain of adenylyl cyclase Rv1625c from Mycobacterium tuberculosis

The Rv1625c gene product is an adenylyl cyclase identified in the genome of Mycobacterium tuberculosis strain H37Rv. It shows sequence similarity to the mammalian nucleotide cyclases and functions as a homodimer, with two substrate-binding sites at the dimer interface. A mutant form of the catalytic domain of this enzyme, K296E/F363R/D365C (KFD→ERC), was overexpressed in Escherichia coli cells in a soluble form. Crystals were obtained using the hanging-drop vapour-diffusion method with PEG 8000 as a precipitant. The protein crystallized in space group P41, with unit-cell parameters a=b=71.25, c=44.51 Å. X-ray diffraction data were collected to a resolution of 3.4 Å and the structure has been solved by the molecular-replacement method using a previously built theoretical model of the protein as the search molecule

Purification, crystallization and preliminary X-ray diffraction analysis of the catalytic domain of adenylyl cyclase Rv1625c from Mycobacterium tuberculosis

The Rv1625c gene product is an adenylyl cyclase identiÆed in the genome of Mycobacterium tuberculosis strain H37Rv. It shows sequence similarity to the mammalian nucleotide cyclases and functions as a homodimer, with two substrate-binding sites at the dimer interface. A mutant form of the catalytic domain of this enzyme, K296E/F363R/D365C (KFD \rightarrow ERC), was overexpressed in Escherichia coli cells in a soluble form. Crystals were obtained using the hanging-drop vapour-diffusion method with PEG 8000 as a precipitant. The protein crystallized in space group $P4_1$, with unit-cell parameters a = b = 71.25, c = 44.51 \AA . X-ray diffraction data were collected to a resolution of 3.4 \AA and the structure has been solved by the molecular-replacement method using a previously built theoretical model of the protein as the search molecule