title: Structure of amidase from Pseudomonas aeruginosa showing a trapped acyl transfer reaction intermediate state.
abstract: Microbial amidases belong to the thiol nitrilases family and have potential biotechnological applications in chemical and pharmaceutical industries as well as in bioremediation. The amidase from Pseudomonas aeruginosa isa6 x 38-kDa enzyme that catalyzes the hydrolysis of a small range of short aliphatic amides. The hereby reported high resolution crystallographic structure shows that each amidase monomer is formed by a globular four-layer alphabetabetaalpha sandwich domain with an additional 81-residue long C-terminal segment. This wraps arm-in-arm with a homologous C-terminal chain of another monomer, producing a strongly packed dimer. In the crystal, the biological active homo-hexameric amidase is built grouping three such dimers around a crystallographic 3-fold axis. The structure also elucidates the structural basis for the enzyme activity, with the nitrilases catalytic triad at the bottom of a 13-A deep, funnel-shaped pocket, accessible from the solvent through a narrow neck with 3-A diameter. An acyl transfer intermediate, resulting from the purification protocol, was found bound to the amidase nucleophilic agent, Cys(166). These results suggest that some pocket defining residues should undergo conformational shifts to allow substrates and products to access and leave the catalytic pocket, for turnover to occur.
authors: Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República, Apartado 127, 2781-901 Oeiras, Portugal.
authors: Jorge
authors: J
authors: Andrade
authors: Amin
authors: A
authors: Karmali
authors: Maria A
authors: MA
authors: Carrondo
authors: Carlos
authors: C
authors: Frazão
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D018360:Crystallography, X-Ray; D019281:Dimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D011550:Pseudomonas aeruginosa / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D000581:Amidohydrolases; C019665:amidase