HALOLYSIN R4, A SERINE PROTEINASE FROM THE HALOPHILIC ARCHAEON HALOFERAX-MEDITERRANEI - GENE CLONING, EXPRESSION AND STRUCTURAL STUDIES

A gene encoding a halophilic serine proteinase, halolysin R4, from a h alophilic archaeon Haloferax mediterranei strain R4 was cloned, its nu cleotide sequence determined, and expressed in Haloferax volcanii WFD1 1. The deduced amino-acid sequence (403 aa in length) showed the highe st similarity to halolysin 172P1, produced by another halophilic archa eon, strain 172P1 (now designated as Natrialba asiatica). Both halolys ins belong to the thermitase branch of class I subtilases, but show lo ng C-terminal extensions of 117 and 123 amino acids, respectively. Rem oval of this 'tail' region from halolysin R4 abolished proteinase acti vity, indicating it provides an essential (but as yet unknown) functio n. Substitution of the two cysteine residues in the C-terminal extensi on with serine decreased enzyme stability in hypotonic solutions: poss ibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).