Preprosubtilisin Carlsberg processing and secretion is blocked after deletion of amino acids 97-101 in the mature part of the enzyme

Abstract

Summary. 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 proteasedeficient 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.

The complete general secretory pathway in gram-negative bacteria.

“…Similar results were obtained in studies on another subtilisin derivative in which four residues had been deleted from the substrate-binding cleft (494). Thus, in this exceptional case, cleavage of the propeptide rather than the signal sequence seems to be required for release from the cytoplasmic membrane.…”

Homology modelling and protein engineering strategy of subtilases, the family of subtilisin-like serine proteinases

“…We propose that substrate specificity may not only be modulated by natural variations in the amino acid sequence of segment 96-104, but also by variations in the length of this exposed loop (VR7). Indeed, deletion of residues 97 -101 in BLSCAR abolishes activity (Schülein et al, 1991) and our own engineering studies of LLSK11 have demonstrated that deletions, insertions and point mutations in this loop can alter specificity and catalytic activity (P.Vos, I.van Alan-Boerrigter, P.Bruinenberg, F.A. Exterkate, M.Nijhuis, W.M.de Vos and R.J.Siezen, in preparation) .…”