Maturation of Fibrinolytic Bacillopeptidase F Involves both Hetero- and Autocatalytic Processes.
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.
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China College of Life Science and Technology, Xinjiang University, Urumuqi, China.
Dongheng
D
Meng
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Meihong
M
Dai
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Bi-Lin
BL
Xu
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Zhong-Shu
ZS
Zhao
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Xiaoliang
X
Liang
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China.
Mingqiu
M
Wang
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China.
Xiao-Feng
XF
Tang
State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan, China tangb@whu.edu.cn.
Bing
B
Tang
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D055162:Biocatalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005342:Fibrinolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D017510:Protein Folding; D046188:Protein Modification, Translational; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012988:Soil Microbiology; D014508:Urea / Q000494:pharmacology
D010455:Peptides; D014508:Urea; D012697:Serine Endopeptidases; C038477:bacillopeptidase F