Expression, purification, crystallization and preliminary crystallographic analysis of a deblocking aminopeptidase from Pyrococcus horikoshii

Acta Crystallographica. Section F, Structural Biology and Crystallization Communications
Sophie PorcieroAlain Roussel

Abstract

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.

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Citations

Dec 21, 2010·Journal of Synchrotron Radiation·Romain TalonEric Girard
Nov 8, 2015·Biochimie·Alexandre AppolaireBruno Franzetti
Oct 30, 2007·Journal of Bioscience and Bioengineering·Hyun Sook LeeSung Gyun Kang
Jun 15, 2011·Bioscience, Biotechnology, and Biochemistry·Baolei JiaGang-Won Cheong
Sep 16, 2006·The Journal of Biological Chemistry·Guy SchoehnBruno Franzetti

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