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 P4(1), with unit-cell parameters a = b = 71.25, c = 44.51 A. X-ray diffraction data were collected to a resolution of 3.4 A and the structure has been solved by the molecular-replacement method using a previously built theoretical model of the protein as the search molecule.
Ketkar
Amit D
AD
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India.
Shenoy
Avinash R
AR
Kesavulu
Muppuru M
MM
Visweswariah
Sandhya S
SS
Suguna
Kaza
K
D000262:Adenylyl Cyclases / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D003460:Crystallization; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011092:Polyethylene Glycols / Q000737:chemistry; D017434:Protein Structure, Tertiary; D014961:X-Ray Diffraction / Q000379:methods*
D011092:Polyethylene Glycols; D000262:Adenylyl Cyclases