Elsevier

Biochimie

Volume 94, Issue 3, March 2012, Pages 798-805
Biochimie

Research paper
Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

https://doi.org/10.1016/j.biochi.2011.11.011Get rights and content
Under an Elsevier user license
open access

Abstract

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the 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 behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.

Highlights

► Nep displays optimal activity and stability in salt-saturated solutions. ► Nep becomes unfolded and aggregated in the absence of salt. ► Native-like form of Nep comprises an elongated molecular structure. ► Low salinity induces a compact conformational state of Nep.

Keywords

Natrialba magadii
Extracellular protease
Halophilism
Structure
Stability
Kinetics

Abbreviations

Nep
Natrialba magadii extracellular protease
DMSO
dimethyl sulfoxide
MCA
7-amino-4-trifluoromethyl coumarin
SKIEs
solvent kinetic isotope effects
CD
circular dichroism
DLS
dynamic light scattering
SAXS
small angle X-ray scattering

Cited by (0)

1

Both authors have contributed equally to this work.