Cloning, expression, purification and preliminary X-ray crystallographic studies of 2-methylisocitrate lyase from Salmonella typhimurium

Abstract

In Salmonella typhimurium, propionate is oxidized to pyruvate via the 2-methylcitric acid cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate, is catalysed by 2-methylisocitrate lyase (EC 4.1.3.30). Methylisocitrate lyase (molecular weight 32 kDa) with a C-terminal polyhistidine affinity tag has been cloned and overexpressed in Escherichia coli and purified and crystallized under different conditions using the hanging-drop vapour-diffusion technique. Crystals belong to the orthogonal space group P2₁2₁2₁, with unit-cell parameters a=63.600, b=100.670, c=204.745 Å. A complete data set to 2.5 Å resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.