TAILIEUCHUNG - Báo cáo khoa học: Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable, active helical form

Nitrilases convert nitriles to the corresponding carboxylic acids and ammo-nia. The nitrilase fromRhodococcus rhodochrousJ1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327, resulting in the formation of active, helical homo-oligomers. | ỊFEBS Journal Post-translational cleavage of recombinantly expressed nitrilase from Rhodococcus rhodochrous J1 yields a stable active helical form R. Ndoria Thuku1 2 Brandon W. Weber2 Arvind Varsani2 and B. Trevor Sewell1 2 1 Department of Biotechnology University of the Western Cape Bellville South Africa 2 Electron Microscope Unit University of Cape Town Rondebosch South Africa Keywords electron microscopy helix IHRSR nitrilase oligomeric form Correspondence B. T. Sewell Electron Microscope Unit University of Cape Town Private Bag Rondebosch 7701 South Africa Fax 272 168 91528 Tel 272 165 02817 E-mail sewell@ Received 23 January 2007 revised 14 February 2007 accepted 20 February 2007 Nitrilases convert nitriles to the corresponding carboxylic acids and ammonia. The nitrilase from Rhodococcus rhodochrous J1 is known to be inactive as a dimer but to become active on oligomerization. The recombinant enzyme undergoes post-translational cleavage at approximately residue 327 resulting in the formation of active helical homo-oligomers. Determining the 3D structure of these helices using electron microscopy followed by fitting the stain envelope with a model based on homology with other members of the nitrilase superfamily enables the interacting surfaces to be identified. This also suggests that the reason for formation of the helices is related to the removal of steric hindrance arising from the 39 C-terminal amino acids from the wild-type protein. The helical form can be generated by expressing only residues 1-327. doi Nitrilases are useful industrial enzymes that convert nitriles to the corresponding carboxylic acids and ammonia. They belong to a superfamily 1 that includes amidases acyl transferases and N-carbamoyl-D-amino acid amidohydrolases and they occur in both prokaryotes and eukaryotes. Their applications include the manufacture of nicotinic acid ibuprofen and acrylic acid and the detoxification of cyanide .

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