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 halophilic archaeon Haloferax mediterranei strain R4 was cloned, its nucleotide sequence determined, and expressed in Haloferax volcanii WFD11. The deduced amino-acid sequence (403 aa in length) showed the highest similarity to halolysin 172P1, produced by another halophilic archaeon, strain 172P1 (now designated as Natrialba asiatica). Both halolysins belong to the thermitase branch of class I subtilases, but show long C-terminal extensions of 117 and 123 amino acids, respectively. Removal of this "tail' region from halolysin R4 abolished proteinase activity, indicating it provides an essential (but as yet unknown) function. Substitution of the two cysteine residues in the C-terminal extension with serine decreased enzyme stability in hypotonic solutions, possibly owing to disruption of potential disulfide bonds or perturbation of calcium binding site(s).
Kamekura
M
M
Noda Institute for Scientific Research, Chiba-ken, Japan.
Seno
Y
Y
Dyall-Smith
M
M
D000595:Amino Acid Sequence; D001105:Archaea / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D012694:Serine; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D012965:Sodium Chloride / Q000494:pharmacology
D017931:DNA Primers; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D012965:Sodium Chloride; D012694:Serine; D012697:Serine Endopeptidases; C088712:halolysin; D003545:Cysteine