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

Abstract

bThe haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I WT ), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M WT ) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitinbinding capacity and mediated the binding of N*-I WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I WT released soluble peptides from SSP faster than did M WT . Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.

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

“…). Such a stepwise maturation process has been described for other linker peptide–containing subtilases, including Tk‐subtilisin (Tanaka et al ., ), protease CDF (Cheng et al ., ), WF146 protease (Zhu et al ., ), and SptC (Zhang et al ., ). For linker peptide‐lacking subtilases such as subtilisins E (Yabuta et al ., ) and BPN′ (Gallagher et al ., ), following autocleavage of the N‐terminal propeptide, the newly liberated N‐terminus of the mature domain moves away from the active site and contributes to the formation of a Ca 2+ ‐binding site (Yabuta 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.…”

“…We previously cloned three halolysin‐encoding genes from the strain J7 genome, designated sptA , sptB (these two genes are arranged in tandem; ), and sptC (). SptA and SptC have been characterized in terms of enzymatic properties and functions of their CTEs (Shi et al ., ; Xu et al ., ; Zhang et al ., ). SptA is stable at high salt concentrations and exhibits higher enzymatic activity in NaCl solution than KCl solution (Shi et al ., ).…”

“…Characterized halolysins include 172P1 from Natrialba asiatica (Kamekura et al ., ), R4 from Haloferax mediterranei (Kamekura et al ., ), SptA and SptC from Natrinema sp. J7 (Shi et al ., ; Zhang et al ., ), and Nep from Nab. magadii (De Castro et al ., ).…”

Alternative Translation Initiation of a Haloarchaeal Serine Protease Transcript Containing Two In-Frame Start Codons

“…DQ137266). The enzymatic properties, maturation, and secretion mechanisms of SptA and SptC have been characterized (25)(26)(27)(28). As the major extracellular protease of Natrinema sp.…”