Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes

Abstract

Bacillopeptidase F (Bpr) is a fibrinolytic serine protease produced by Bacillus subtilis. Its precursor is composed of a signal peptide, an N-terminal propeptide, a catalytic domain, and a long C-terminal extension (CTE). Several active forms of Bpr have been previously reported, but little is known about the maturation of this enzyme. Here, a gene encoding a Bpr (BprL) was cloned from B. subtilis LZW and expressed in B. subtilis WB700, and three fibrinolytic mature forms with apparent molecular masses of 45, 75, and 85 kDa were identified in the culture supernatant. After treatment with urea, the 75-kDa mature form had the same molecular mass as the 85-kDa mature form, from which we infer that they adopt different conformations. Mutational analysis revealed that while the 85-kDa mature form is generated via heterocatalytic processing of a BprL proform by an unidentified protease of B. subtilis, the production of the 75-and 45-kDa mature forms involves both hetero-and autocatalytic events. From in vitro analysis of BprL and its sequential C-terminal truncation variants, it appears that partial removal of the CTE is required for the initiation of autoprocessing of the N-terminal propeptide, which is composed of a core domain (N*) and a 15-residue linker peptide, thereby yielding the 45-kDa mature form. These data suggest that the differential processing of BprL, either heterocatalytically or autocatalytically, leads to the formation of multiple mature forms with different molecular masses or conformations.

Natto extract inhibits infection caused by the Aujeszky's disease virus in mice

“…Bacillus subtilis produces intracellular and extracellular serine proteases. [16][17][18][19][20][21][22] Extracellular serine proteases are secreted outside the bacterium and function as degradative proteases required to acquire proteins from the environment as a source of nutrients. 22 One or more serine proteases in Fraction No.…”

“…However, the extra bacterial serine proteases produced by Bacillus natto have a molecular weight of more than 20 kDa. [16][17][18][19][20][21][22] Therefore, it is necessary to examine the proteolytic agent(s) involved in ADV inactivation by natto extract, considering the possibility that they are not proteases. Nakamura et al 23 revealed the presence of a small peptide with protease activity.…”

“…10 may be involved in ADV inactivation. However, the extra bacterial serine proteases produced by Bacillus natto have a molecular weight of more than 20 kDa 16–22 . Therefore, it is necessary to examine the proteolytic agent(s) involved in ADV inactivation by natto extract, considering the possibility that they are not proteases.…”

Biodegradation of Pine Processionary Caterpillar Silk Is Mediated by Elastase- and Subtilisin-like Proteases

“…Among the best candidates we should mention the two bacillopeptidases F detected (proteins 2 and 3 in Table S1—Supplementary Material ). Bacillopeptidases F are extracellular fibrinolytic serine proteases [ 31 ] belonging to the superfamily of subtilisin-like serine proteases. These enzymes are expressed at the beginning of the stationary phase in B. subtilis .…”

Microbial Fibrinolytic Enzymes as Anti-Thrombotics: Production, Characterisation and Prodigious Biopharmaceutical Applications

“…In biotechnological applications, the non-pathogenicity of B. subtilis and its ability of exuding valuable extracellular proteins in growth medium signify its suitability as an efficient host for the expression of foreign proteins [111]. Gene encoding bacillopeptidase F was cloned from B. subtilis LZW, expressed in B. subtilis WB700 and the catalytic mechanism of proteins along with the activity of C-terminal truncation variants in sustaining enzymatic activity were interpreted [112]. A significant increase in fibrinolytic activity (80-200 urokinase U/mL) was observed when the gene expression of subtilisin DFE in B. subtilis was mediated by a promoter of α-amylase gene from B. amyloliquefaciens DC-4 [113].…”