The Maturation Of Halolysin Sptc And The Function Of Its Chitin-binding Domain

Chitin is the major constituent of the shells of crustaceans such as crabs and shrimps, the exoskeletons of insects, and the cell walls of many fungi. A large number of microorganisms produce enzymes including chitinases, ?-N-acetylglucosaminidases, and proteases to digest chitin-containing biomass, and utilize the degradation products as carbon source and energy source for growth. The chitin catabolism pathway in bacteria and eukarya has been well characterized, but the chitin catabolism pathway in haloarchaea has been established only in Haloferax mediterranei. The haloarchaeon Natrinema sp. J7-2was isolated from a salt mine in Hubei province, and its genome harbors a chitin metabolism-related gene cluster containing a halolysin gene, sptC. In contrast to the C-terminal extension ?CTE? of other halolysins, the CTE of SptC contains a polycystic kidney disease domain ?PkdD? and a chitin-binding domain ?ChBD?. In the present study, we investigated the maturation process of SptC and its role in the chitin metabolism of Natrinema sp. J7-2.Firstly, genomic analysis of Natrinema sp. J7-2revealed the presence of a chitin metabolism-related gene cluster ?NJ7G₂696-2707? that encodes a chitin-binding protein ?Cbp?, SptC, three chitinases ?Chil, Chi2, and Chi3?, the sugar ABC transporter system and intracellular glycoside hydrolase. A genomic survey of sequenced haloarchaea showed that this gene cluster is highly conserved in Natrinema species. SptC, Cbp, Chil, Chi2and Chi3of Natrinema sp. J7-2share two domains, a PkdD and a ChBD. The PkdDs or ChBDs of these five proteins share significant sequence identity, suggesting that an evolutionary relationship exists among these proteins. Meanwhile, strain J7-2has the ability to degrade chitin and utilizes the degradation product as the sole carbon source.Secondly, we investigated the maturation process of recombinant SptC. Several deletion mutants of the enzyme were constructed to probe possible roles of the N-terminal propeptide, the PkdD and the ChBD in the maturation process and enzymatic properties of SptC. The autocatalytic maturation of SptC occurs in a stepwise manner. First, the core domain of the N-terminal propeptide ?N*? is cis-processed to yield an autoprocessed complex ?N*-IWT?. Next, the five-residue linker peptide and the C-terminal ChBD are trans-processed from the intermediate (IWT and N*is degraded, leading to generation of the active mature form composed of the catalytic domain and the PkdD. The N-terminal propeptide deletion mutant SptCAN was unable to mature, implying that the N-terminal propeptide acts as an intramolecular chaperone to assist enzyme folding. Additionally, the CTE is not necessary for enzyme folding and activaion, but enzyme activation is facilitated by the ChBD and the PkdD. Meanwhile, all four mature forms showed optimum azocaseinolytic activity at3-3.5M NaCl, demonstrating salt-dependent stability. The PkdD of SptC does not confer extra stability on the enzyme but instead contributes to the enzymatic activity of the mature form.Thirdly, we investigated the chitin-related functions of SptC. The ChBD exhibited salt-dependent chitin-binding capacity and mediated the binding of N*-IWT to chitin. SptC showed no significant binding capacity to cellulose and chitosan. Meanwhile, activation of all four autoprocessed complexes was significantly accelerated in the presence of chitin or cellulose. ChBD-mediated chitin binding of N*-IWT or N*_I?PkdD enhances the chance of interaction between them, and leads to an increase in the local enzyme concentration, thus further accelerating the maturation of SptC and SptCAPkdD. In addition, SptA, pyrolysin, S41, sphericase and WF146protease, which are the homologs of SptC without the ChBD, were used to investigate the mechanism of polysaccharide-accelerated protease activation. Our results revealed that SptA, pyrolysin, S41, sphericase and WF146protease showed no binding capacity to chitin, cellulose and chitosan. Nevertheless, the maturation of WF146protease was accelerated in the presence of chitin or cellulose to some degree. SptCACTE, SptCAChBD and WF146protease displayed similar polysaccharide-accelerated activation profiles. These results demonstrate that the polysaccharide-accelerated protease activation is not exclusively dependent on the presence of ChBD, but the amino acid sequence and structure of proteases.Finally, our results also demonstrate that SptC is capable of removing proteins from chitin-containing biomass such as shrimp shell powder ?SSP? at high salt concentrations. Interestingly, the N*-IWT-treated sample showed a faster release of soluble peptides than the MWT-treated sample. It is very likely that ChBD-mediated binding of N*-IWT to SSP not only promotes enzyme activation but also increases the local concentration of active enzyme around the SSP, thus enhancing deproteinization. These results demonstrate that halolysin SptC plays an important role in the chitinolytic system of Natrinema sp. J7-2and the deproteinization of chitin-containing biomass.