Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme.
During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, Mr = 42 kDa) was not processed to the mature form (Mr = 30 kDa) and was not released into the medium by a protease-deficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants from B. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0-3 h after onset of stationary phase) of a processing intermediate (P38c, Mr = 38 kDa) of this protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.
Schülein
R
R
Institut für Genetik und Mikrobiologie, Universität Würzburg, FRG.
Kreft
J
J
Gonski
S
S
Goebel
W
W
D001407:Bacillus / Q000201:enzymology / Q000235:genetics*; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004792:Enzyme Precursors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015183:Restriction Mapping; D013381:Subtilisins / Q000235:genetics* / Q000302:isolation & purification
D004792:Enzyme Precursors; D013381:Subtilisins; C049752:preprosubtilisin