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

M Asuncion, J N Barlow, J Pollard, A G Staines, Stephen McMahon, W Blankenfeldt, David Gani, Jim Naismith

Research output: Contribution to journalArticlepeer-review

3 Citations (Scopus)

Abstract

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 acid 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 step of the catabolic pathway for glutamic acid in Clostridium tetanomorphum. The reverse reaction is of particular interest because the addition of ammonia 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 enzyme have been obtained by sitting-drop vapour diffusion. Two native data sets have been collected, one in-house on a rotating-anode generator to 3.2 Angstrom and one at the European Synchrotron Radiation Facility to 2.0 Angstrom. A 2.1 Angstrom data set has been collected on a crystal of selenomethionine 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 with 42% solvent. A self-rotation function indicates the presence of a twofold axis, consistent with a biological dimer.

Original languageEnglish
Pages (from-to)731-733
Number of pages3
JournalActa Crystallographica. Section D, Biological crystallography
Volume57
Issue number5
DOIs
Publication statusPublished - May 2001

Keywords

  • ASPARTIC ACIDS
  • FUMARIC ACIDS
  • METHYLASPARTASE
  • ELIMINATION
  • MECHANISM
  • AMMONIA
  • PROTEINS

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