Involvement of NH2-terminal pro-sequence in the production of active aqualysin I (a thermophilic serine protease) in Escherichia coli.
Aqualysin I is a heat-stable subtilisin-type protease produced by Thermus aquaticus YT-1. The precursor of aqualysin I consists of four domains: an NH2-terminal signal peptide, an NH2-terminal pro-sequence, a protease domain, and a COOH-terminal pro-sequence. In Escherichia coli cells harboring recombinant plasmid carrying the aqualysin I gene, proteolytic activity is obtained on treatment at 65 degrees C and mature enzyme is detected. In the case of mutant genes containing partial deletions in the NH2-terminal pro-sequence, no proteolytic activity was detected and the precursor protein was found to be unstable in E. coli. These results indicate that the NH2-terminal pro-sequence is required to produce the active enzyme by stabilizing the precursor structure. Amino acid substitutions in the conserved sequence of the NH2-terminal pro-sequence found among subtilisin-type proteases made the processing faster compared with the wild type.
Department of Agricultural Chemistry, University of Tokyo, Japan.
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YC
Lee
Y
Y
Miyata
I
I
Terada
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Ohta
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Matsuzawa
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D011498:Protein Precursors / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D013824:Thermus / Q000201:enzymology*
D000596:Amino Acids; D011498:Protein Precursors; D012697:Serine Endopeptidases; C055102:aqualysin I