CONICET | Buscador de Institutos y Recursos Humanos

Archaeal halophilic subtilases, so called halolysins, are modular enzymes consisting of a subtilisin-like catalytic domain linked to a C-terminal domain with unknown function. While their adaptation to halophilic environments has been extensively described, the molecular mechanism involved in their catalytic activity under highly saline conditions is still elusive. Nep is a halolysin produced by the haloalkaliphilic archaeon Natrialba magadii ATCC 43099 that exhibits optimum activity and stability in salt-saturated solutions. In this work, the effect of salt on the functional and structural behavior of Nep has been investigated. In absence of salt, Nep becomes unfolded and aggregates, leading to loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M NaCl, Nep behaves as monomers in solution and its enzymatic activity displayed nonlinear concave-up dependence with salt concentration. Although transition from a high to a low saline environment (3 M to 1 M NaCl) did not affect the secondary structure content of Nep, it diminishes the enzyme stability and its two-domain structural arrangement is drastically changed from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. While solvent kinetic isotopic effect (SKIE) data indicate that Nep shares with canonical serine-peptidases the same rate limiting steps, thermodynamic analysis of Nep peptide hydrolysis suggests that a larger reorganization of Nep occurs during catalysis in moderate salt environments. All these data correlate functional and structural behavior of Nep providing a better understanding of how catalysis can be affected by stability and conformational changes in halolysins.