Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium.
In the cell, propionate is mainly formed during beta-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that L-threonine is non-oxidatively cleaved to propionate via 2-ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.-). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 111.47, c = 66.52 A. A complete data set to 2.2 A resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.
Simanshu
Dhirendra K
DK
Molecular Biophysics Unit, Indian Institute of Science, Bangalore, India.
Murthy
M R N
MR
D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000201:enzymology; D017851:Phosphotransferases (Carboxyl Group Acceptor) / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011422:Propionates / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology*; D013912:Threonine / Q000378:metabolism; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D017931:DNA Primers; D011422:Propionates; D011994:Recombinant Proteins; D013912:Threonine; D017851:Phosphotransferases (Carboxyl Group Acceptor)