Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.
The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.
Arnórsdottir
Jóhanna
J
Institute of Biology, University of Iceland; and Department of Biochemistry, Science Institute, University of Iceland, Reykjavik, Iceland.
Smáradóttir
Rúna B
RB
Magnússon
Olafur Th
OT
Thorbjarnardóttir
Sigrídur H
SH
Eggertsson
Gudmundur
G
Kristjánsson
Magnús M
MM
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003080:Cold Temperature; D004247:DNA / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D019286:Endopeptidase K / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D008024:Ligands; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics*; D020860:Subtilisin / Q000737:chemistry* / Q000235:genetics*; D014733:Vibrio / Q000201:enzymology*
D008024:Ligands; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D004247:DNA; D012697:Serine Endopeptidases; D020860:Subtilisin; D019286:Endopeptidase K; D019791:Guanidine