Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

Zhu, Hui; Bin, Xu; Liang, Xiaoliang; Yang, Yiran; Tang, Xiaofeng; Tang, Bing

doi:10.1074/jbc.m113.498774

Cited by 15 publications

(17 citation statements)

References 34 publications

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“…5C and D, ϪM). We found that the cisprocessed N* of BprL⌬C912, BprL⌬C912-S258C, BprL⌬C678, or BprL⌬C678-S258C coeluted with Ia or Ia= from an Ni 2ϩ -charged column (data not shown), implying that the cleaved N* was still bound to Ia or Ia= to form an autoprocessed complex (N*-Ia or N*-Ia=), as has been reported for other subtilisins (28,29). In the presence of the active 45-kDa mature enzyme, either N*-Ia or N*-Ia= was trans processed into the 45-kDa mature form (Fig.…”

Section: Fig 4 In Vitro Processing Of Bprl and Its Variants (A To C)supporting

confidence: 66%

“…It is known that replacing the catalytic residue Ser with Ala abolishes the activity of subtilases but does not affect enzyme folding (28,29). To investigate whether the multiple proteolytic processing of BprL occurs auto-or heterocatalytically, the culture supernatant of B. subtilis WB700(pBE2-bprLS258A) producing the active-site variant BprLS258A was subjected to SDS-PAGE and immunoblot analyses.…”

Section: Resultsmentioning

confidence: 99%

“…In subtilases, the replacement of the catalytic residue Ser with Cys greatly reduces enzyme activity but does not eliminate its autoprocessing activity, so that the maturation process is blocked after the initial cis processing of the N-terminal propeptide (28,29). Similarly, the variant BprL⌬C912-S258C or BprL⌬C678-S258C itself was converted into the intermediate Ia or Ia= by cis processing of N* but could not be further processed into the 45-kDa mature form (Fig.…”

Section: Fig 4 In Vitro Processing Of Bprl and Its Variants (A To C)mentioning

confidence: 99%

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

Meng

Dai

Bin

et al. 2016

Appl Environ Microbiol

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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.

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Section: Fig 4 In Vitro Processing Of Bprl and Its Variants (A To C)supporting

confidence: 66%

Section: Resultsmentioning

confidence: 99%

Section: Fig 4 In Vitro Processing Of Bprl and Its Variants (A To C)mentioning

confidence: 99%

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

Meng

Dai

Bin

et al. 2016

Appl Environ Microbiol

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“…The major difference between the maturations of the two halolysins is that the Nep precursor can be trans-processed into its mature form by active Nep, but the SptC precursor is degraded by active mature SptC. Generation of the mature enzyme via transprocessing of the N-terminal propeptide of the precursor or the proform, either auto-or heterocatalytically, has also been described for other subtilases (27,(35)(36)(37)(38). In the precursor of the WF146 protease from thermophilic Bacillus sp.…”

Section: Discussionmentioning

confidence: 99%

“…In the precursor of the WF146 protease from thermophilic Bacillus sp. WF146, a 12-residue linker peptide between the core domain of the N-terminal propeptide and the catalytic domain comprises a proteolytically sensitive region, but the catalytic domain adopts a stable conformation that is resistant to proteolysis, allowing the trans-processing reaction to occur auto-and heterocatalytically (38). Although the SptC precursor contains a five-residue linker peptide, its catalytic domain is susceptible to proteolysis.…”

Section: Discussionmentioning

confidence: 99%

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

Zhang

Wang

et al. 2014

Appl Environ Microbiol

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bThe haloarchaeon Natrinema sp. strain J7-2 has the ability to degrade chitin, and its genome harbors a chitin metabolism-related gene cluster that contains a halolysin gene, sptC. The sptC gene encodes a precursor composed of a signal peptide, an N-terminal propeptide consisting of a core domain (N*) and a linker peptide, a subtilisin-like catalytic domain, a polycystic kidney disease domain (PkdD), and a chitin-binding domain (ChBD). Here we report that the autocatalytic maturation of SptC is initiated by cis-processing of N* to yield an autoprocessed complex (N*-I WT ), followed by trans-processing/degradation of the linker peptide, the ChBD, and N*. The resulting mature form (M WT ) containing the catalytic domain and the PkdD showed optimum azocaseinolytic activity at 3 to 3.5 M NaCl, demonstrating salt-dependent stability. Deletion analysis revealed that the PkdD did not confer extra stability on the enzyme but did contribute to enzymatic activity. The ChBD exhibited salt-dependent chitinbinding capacity and mediated the binding of N*-I WT to chitin. ChBD-mediated chitin binding enhances SptC maturation by promoting activation of the autoprocessed complex. Our results also demonstrate that SptC is capable of removing proteins from shrimp shell powder (SSP) at high salt concentrations. Interestingly, N*-I WT released soluble peptides from SSP faster than did M WT . Most likely, ChBD-mediated binding of the autoprocessed complex to chitin in SSP not only accelerates enzyme activation but also facilitates the deproteinization process by increasing the local protease concentration around the substrate. By virtue of these properties, SptC is highly attractive for use in preparation of chitin from chitin-containing biomass.

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Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures

Liu

Zhao

Ren

et al. 2016

Appl Microbiol Biotechnol

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Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by α-carboxyl groups of Glu and Asp residues. We cloned the gene for a thermophilic GSE (designated TS-GSE) from Thermoactinomyces sp. CDF. A proform of TS-GSE that contained a 61-amino acid N-terminal propeptide and a 218-amino acid mature domain was produced in Escherichia coli. We found that the proform possessed two processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be autoprocessed at the Glu-Ser bond to directly generate the mature enzyme. It could also be autoprocessed at the Glu-Lys bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by either active TS-GSE or heterogeneous proteases. Deletion analysis revealed that the N-terminal propeptide is important for the correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain. The mature TS-GSE displayed a temperature optimum of 85 °C and retained approximately 90 % of its original activity after incubation at 70 °C for 6 h, representing the most thermostable GSE reported to date. Mutational analysis suggested that the disulfide bonds Cys-Cys and Cys-Cys cumulatively contributed to the thermostability of TS-GSE.

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Molecular Basis for Auto- and Hetero-catalytic Maturation of a Thermostable Subtilase from Thermophilic Bacillus sp. WF146

Cited by 15 publications

References 34 publications

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

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

Chitin Accelerates Activation of a Novel Haloarchaeal Serine Protease That Deproteinizes Chitin-Containing Biomass

Autocatalytic activation of a thermostable glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures

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