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

Abstract

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 maturation mechanism of halolysins has not been addressed. The halolysin Nep is secreted by the alkaliphilic haloarchaeon Natrialba magadii, and the recombinant active enzyme has been synthesized in Haloferax volcanii. Nep contains an N-terminal signal peptide with the typical Tat consensus 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 and maturation of halolysins by using genetic and biochemical approaches. Mutant variants of Nep were constructed by site-directed mutagenesis and expressed in H. volcanii, which were then analyzed by protease activity and Western 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 extracellularly 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 and maturation of a Tat-dependent halophilic subtilase. Subtilisin-like proteases (subtilases) are a functionally diverse superfamily of serine proteases produced by prokaryotes and eukaryotes that include both intra-and extracellular enzymes (30). These proteases serve in a number of biotechnological applications (11,18,24); thus, subtilases from extremophiles represent an attractive resource for both basic and applied science.Secretory subtilases are synthesized as prepro-enzymes consisting of a signal peptide (SP) responsible for secretion, N-terminal and/or C-terminal propeptides, and the catalytic domain (S8 family). Bacterial prepro-subtilisins are processed by removal of the pre-and/or propeptides to render the active extracellular enzymes (30). Comparatively less is known regarding how extracellular proteases are transported through the archaeal membrane and processed into fully active enzymes. In this context, the maturation mechanism of the Sec-dependent subtilisin-like proteases secreted by the hyperthermophilic archaeon Thermococcus kodaraensis have been extensively characterized (8, 33). Many halophilic bacteria and archaea produce extracellular proteases, however, there is limited information on the pathways used by halophiles to deliver secretory proteases (27,28), and the mechanism that leads to enzyme activation has not been examined.Most extracellular and membrane anchored proteins are expor...

Secretion of Tat‐dependent halolysin SptA capable of autocatalytic activation and its relation to haloarchaeal growth

“…Each of these halolysins is synthesized as a precursor containing a Tat signal peptide, an N‐terminal propeptide, a subtilisin‐like catalytic domain, and a C‐terminal extension (CTE). Mutation analyses of the Tat signal peptides of SptA and Nep have shown that these halolysins are Tat‐dependent substrates (Shi et al ., ; Ruiz et al ., ). Nep and SptC reportedly mature autocatalytically (Ruiz et al ., ; Zhang et al ., ).…”

“…Mutation analyses of the Tat signal peptides of SptA and Nep have shown that these halolysins are Tat‐dependent substrates (Shi et al ., ; Ruiz et al ., ). Nep and SptC reportedly mature autocatalytically (Ruiz et al ., ; Zhang et al ., ). These findings raise interesting questions about whether Tat‐dependent halolysins can be prematurely activated before translocation across the cytoplasmic membrane in a folded state and how haloarchaea minimize the risk of premature activation of halolysins and associated proteolytic damage to cellular proteins.…”

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

“…Although the PkdD of SptC and the CTEs of other halolysins (e.g., SptA and Nep) show no significant homology, they are predicted to adopt a similar ␤-jelly roll-like structure. The importance of the CTE to enzymatic activity has been reported for halolysins R4 (7), SptA (11), and Nep (10). The CTE of SptA assists enzymatic activity toward protein substrates rather than smallpeptide substrates by facilitating the binding of protein substrates for catalysis (11).…”

“…The mature forms of halolysins are composed of not only a highly conserved catalytic domain (ϳ60 to 80% identity) but also a CTE. The CTEs of the above-mentioned halolysins also share high sequence identity (ϳ40 to 70%) and are reportedly important for the stability and activity of R4 (7), SptA (11), and Nep (10). The attachment of an extension to the C terminus of the catalytic domain appears to be a common strategy employed by halolysins to adapt to hypersaline environments.…”

“…Each of these extracellular enzymes is synthesized as a precursor containing a signal peptide, an N-terminal propeptide, a subtilisin-like catalytic domain, and a C-terminal extension (CTE). Like bacterial subtilases, the signal peptide of halolysin precursors is removed after secretion by a signal peptidase to generate a proform, and then the N-terminal propeptide of the proform is autoprocessed to yield the active mature halolysin (10). The mature forms of halolysins are composed of not only a highly conserved catalytic domain (ϳ60 to 80% identity) but also a CTE.…”