Overexpression, purification, crystallization and data collection of 3-methylaspartase from Clostridium tetanomorphum

3-Methylaspartase (E.C. 4.3.1.2) catalyses the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic aci d as well as a slower syn elimination from the (2S,3R)-epimer, L-erythro-3- methylaspartic acid. The anti-elimination reaction occurs in the second ste p of the catabolic pathway for glutamic acid in Clostridium tetanomorphum. The reverse reaction is of particular interest because the addition of ammo nia to substituted fumaric acids is highly stereoselective and gives highly functionalized amino acids. The mechanism of the transformation is unusual and of considerable interest. 3-Methylaspartase from C. tetanomorphum has been overexpressed and purified from Escherichia coli. Crystals of the enzy me have been obtained by sitting-drop vapour diffusion. Two native data set s have been collected, one in-house on a rotating-anode generator to 3.2 An gstrom and one at the European Synchrotron Radiation Facility to 2.0 Angstr om. A 2.1 Angstrom data set has been collected on a crystal of selenomethio nine protein. Combining the data sets identify the space group as P2(1)2(1) 2, with unit-cell parameters a = 110.3, b = 109.9, c = 67.2 Angstrom, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers wit h 42% solvent. A self-rotation function indicates the presence of a twofold axis, consistent with a biological dimer.