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

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