Expression, purification, crystallization and preliminary crystallographic analysis of a deblocking aminopeptidase from Pyrococcus horikoshii.
The deblocking aminopeptidase (DAP) of Pyrococcus horikoshii is a hyperthermophilic exoprotease that cleaves the N-terminal amino acid of peptide substrates with a putative deblocking activity for acylated peptides. DAP has been found to be homologous to a tetrahedral aminopeptidase from the halophilic Haloarcula marismortui. The latter enzyme is a dodecameric complex and has been revealed to be a self-compartmentalized protease whose central cavity harbouring the catalytic site is accessible through several channels of different size, unlike all other known proteolytic complexes. Three paralogues of DAP have been identified in P. horikoshii, with about 40% identity between them. Each of them has been overexpressed in Escherichia coli, purified and crystallized in the native and selenomethionine-substituted states. The results indicate that they form two kinds of assemblies, of 12 and of 24 subunits, with a molecular weight of approximately 400 and approximately 800 kDa, respectively. Crystals of the different variants of DAP and in their different oligomeric states diffract up to a resolution of 3 A.
Porciero
Sophie
S
Architecture et Fonction des Macromolécules Biologiques, UMR 6098 CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
Receveur-Bréchot
Véronique
V
Mori
Kazushige
K
Franzetti
Bruno
B
Roussel
Alain
A
D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology; D008970:Molecular Weight; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification; D043902:Pyrococcus horikoshii / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014961:X-Ray Diffraction
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D000626:Aminopeptidases