Autocatalytic Maturation of the Tat-Dependent Halophilic Subtilase Nep Produced by the Archaeon Natrialba magadii

• DOI: 10.1128/jb.06792-11
• Publication Date: 2012-05-12T05:22:29Z
• Authors: Diego M. Ruiz; Roberto A. Paggi; Maria I. Giménez; Rosana E. De Castro
• Keywords: Subtilisin; Haloarchaea; Haloferax volcanii; Signal peptidase; Protein precursor; Proteolysis; Catalytic triad

ABSTRACT Halolysins are subtilisin-like extracellular proteases produced by haloarchaea that possess unique protein domains and salt dependent for structural integrity functionality. In contrast to bacterial subtilases, the maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted alkaliphilic haloarchaeon Natrialba magadii , recombinant active enzyme synthesized in Haloferax volcanii . contains an N-terminal signal peptide with typical Tat consensus motif (G RR SVL), propeptide, protease domain, a C-terminal domain. this study, we used as model examine secretion using genetic biochemical approaches. Mutant variants were constructed site-directed mutagenesis expressed H. which then analyzed activity Western blotting. dependence was demonstrated RR/KK containing double lysine (KK) place twin arginines (RR), remained cell associated undetectable. High-molecular-mass polypeptides without detected extracellularly S/A variant, catalytic serine 352 had changed alanine, indicating needed precursor processing activation. NSN 1-2 modification two potential cleavage sites peptidase I (ASA) efficiently processed activated. This study examined first time Tat-dependent halophilic subtilase.