Functional roles of C-terminal extension (CTE) of salt-dependent peptidase activity of the Natrialba magadii extracellular protease (NEP).
Marem, A., Okamoto, D. N., Oliveira, L. C. G., Ruiz, D. M., Paggi, R. A., Kondo, M. Y., Gouvea, I. E., Juliano, M. A., de Castro, R. E., Juliano, L. and Icimoto, M. Y.
Departamento de Biofisica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, Brazil.
Instituto de Investigaciones Biotecnologicas de Chascomus, Universidad Nacional de San Martin, Chascomus, Argentina.
Instituto de Investigaciones Biologicas (IIB), Universidad Nacional de Mar del Plata, Mar del Plata, Argentina.
Departamento de Biofisica, Escola Paulista de Medicina, Universidade Federal de Sao Paulo, Sao Paulo, Brazil. Electronic address: icimoto@unifesp.br.
Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon Natrialba magadii. Many extracellular proteases have been characterized from archaea to bacteria as adapted to hypersaline environments retaining function and stability until 4.0M NaCl. As observed in other secreted halolysins, this stability can be related to the presence of a C-terminal extension (CTE) sequence. In the present work, we compared the biochemical properties of recombinant Nep protease with the truncated form at the 134 amino acids CTE (NepCTE), that was more active in 4M NaCl than the non-truncated wild type enzyme. Comparable to the wild type, NepCTE protease is irreversibly inactivated at low salt solutions. The substrate specificity of the truncated NepCTE was similar to that of wild type form as demonstrated by a combinatorial library of FRET substrates. The enzyme stability, the effect of different salts and the thermodynamics assays using different lengths of substrates demonstrated similarities between the two forms. Altogether, these data provide further information on the stability and structural determinants of halolysins under different salinities, especially concerning the enzymatic behavior.
International Journal of Biological Macromolecules 113: 1134-1141 (2018)