Processing And Maturation Mechanism Of Bacillopeptidase F From Bacillus Subtilis LZW

Bacillus subtilis is known to produce many extracellular proteases including bacillopeptidase F (Bpr). Bpr is of great interest in the pharmaceutical area due to its fibrinolytic activity. In previous studies, several active forms of BprL with molecular masses ranging from 33 kDa to 90 kDa have been described. However, the reason for the multiplicity of active forms of Bpr remains unclear. To solve this problem, it is required to investigate the processing and maturation mechanisms of Bpr. Furthermore, the elucidation of maturation mechanism of Bpr will be helpful for the preparation of active Bpr and the application of this enzyme in the pharmaceutical area. Bacillus subtilis LZW was isolated from soil, and was able to produce extracellular proteases. In this study, the gene (bprL) encoding bacillopeptidase F (BprL) was cloned from this bacterium and the processing and maturation mechanisms of this enzyme was investigated.First, the fibrinolytic activity of strain LZW was confirmed by the formation of clear halos around the colonies of this bacterium grown on LB agar plate containing fibrin. Based on the gene sequence of Bpr from B. subtilis 168, the gene bprL was amplified from the genome of strain LZW by PCR, and was sequenced. The gene bprL encodes the precursor of BprL (1433 residues), which is composed of a signal peptide (30 residues), an N-terminal propeptide (164 residues), a catalytic domain (327 residues), and a C-terminal sequence (912 residues). BprL shares 98%, and 97% amino acid sequence identities with the precursors of Bprs from B. subtilis natto and B. subtilis 168, respectively, and the non-conserved residues are mainly located within the C-terminal sequence. In addition, the ORF of bprL is preceded by a DegU-dependent promoter and is followed by a putative transcription termination site.By using B. subtilis WB700 as the host, bprL was expressed and the in vivo processing and maturation of recombinant BprL was investigated. Deletion analysis demonstrated that the upstream sequence (contains DegU-dependent promoter and RBS) of bprL played an important role in the expression of this gene. The SDS-PAGE analysis of the culture supernatant revealed that there were four major BprL-derived proteins with apparent molecular masses of 85,75,45, and 33 kDa, respectively. The Western-blot analysis revealed that the 85 and 75 kDa proteins contained both the catalytic domain and partial C-terminal sequence; the 45 kDa protein contained only the catalytic domain; the 33 kDa protein was the degradation product of the C-terminal sequence. The results of activity assay showed that the 45 kDa protein possessed proteolytic and fibrinolytic activities, and the 85 and/or 75 kDa proteins were also active. The active-site variant of BprL (BprL-S258A) was also constructed and produced in B. subtilis WB700. It was found that BprL-S258A was processed into the 85 kDa protein, but not the 75,45, and 33 kDa proteins. These results suggest that the 75,45, and 33 kDa proteins were autoprocessed products of BprL, while the 85 kDa protein was the hetero-processed product. In addition, the C-terminal sequence deletion variant BprL A 912 could be processed into the 45 kDa protein in B. subtilis WB700, indicating that the 45 kDa protein was the smallest mature form of BprL. Therefore, the in vivo maturation of BprL involves both autoprocessing and heter-processing steps, thus generating multiple mature forms.BprL and its sequential C-terminal truncation variants were also produced in Escherichia coli, and the recombinant proteins were subjected to in vitro renaturation and processing analyses. It was found that the full-length BprL was unable to be converted into active mature forms, while the C-terminal truncation variants BprL?678 and BprLA912 could be processed into the 45 kDa mature forms. This indicates that the C-terminal sequence of BprL contributes to maintain the enzyme in an inactive state, and thus may prevent enzyme premature, which is harmful to cells. Meanwhile, these results also suggest that hetero-processing of partial C-terminal sequence is required for the initiation of autocatalytic maturation of BprL. In addition, we found that the N-terminal propeptide of BprL was removed stepwisely. First, the core domain of the N-terminal propeptide (N*) was cis-processed, mediated by the active-site of the enzyme its self. Then, the remaining N-terminal propeptide fragment (linker peptide) was trans-processed. Furthermore, the modification of the sequence around the cis-processing site Lys(-16)-Ser(-15) of BprL by replacing the residue Phe(-17) with Tyr could accelerate the maturation of this enzyme.