An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

Ono, Toshio; Nemoto, Takayuki; Shimoyama, Yu; Kimura, Shigenobu; Ohara‐Nemoto, Yuko

doi:10.1016/j.ab.2008.06.022

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…This part might be accidentally tagged as a result of a T-to-G mutation from the stop codon (TGA) into Gly 711 (GGA) in the PeDPP7 gene. Similarly, a glutamic acid-specific V8 protease, GluV8, belonging to the S46 family was found to be tagged with the C-terminal 48 amino acid residues, which have been shown to be non-essential for the activity [35] , [36] . The characteristic GluV8 tag is 12 repeats of the triplet Pro-Asp/Asn-Asn [37] .…”

Section: Discussionmentioning

confidence: 99%

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

et al. 2014

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Dipeptidyl peptidases (DPPs) that liberate dipeptides from the N-terminal end of oligopeptides are crucial for the growth of Porphyromonas species, anaerobic asaccharolytic gram negative rods that utilize amino acids as energy sources. Porphyromonas endodontalis is a causative agent of periapical lesions with acute symptoms and Asp/Glu-specific DPP11 has been solely characterized in this organism. In this study, we identified and characterized two P. endodontalis DPPs, DPP5 and DPP7. Cell-associated DPP activity toward Lys-Ala-4-methylcoumaryl-7-amide (MCA) was prominent in P. endodontalis ATCC 35406 as compared with the Porphyromonas gingivalis strains ATCC 33277, 16-1, HW24D1, ATCC 49417, W83, W50, and HNA99. The level of hydrolysis of Leu-Asp-MCA by DPP11, Gly-Pro-MCA by DPP4, and Met-Leu-MCA was also higher than in the P. gingivalis strains. MER236725 and MER278904 are P. endodontalis proteins belong to the S9- and S46-family peptidases, respectively. Recombinant MER236725 exhibited enzymatic properties including substrate specificity, and salt- and pH-dependence similar to P. gingivalis DPP5 belonging to the S9 family. However, the k cat/K m figure (194 µM−1·sec−1) for the most potent substrate (Lys-Ala-MCA) was 18.4-fold higher as compared to the P. gingivalis entity (10.5 µM−1·sec−1). In addition, P. endodontalis DPP5 mRNA and protein contents were increased several fold as compared with those in P. gingivalis. Recombinant MER278904 preferentially hydrolyzed Met-Leu-MCA and exhibited a substrate specificity similar to P. gingivalis DPP7 belonging to the S46 family. In accord with the deduced molecular mass of 818 amino acids, a 105-kDa band was immunologically detected, indicating that P. endodontalis DPP7 is an exceptionally large molecule in the DPP7/DPP11/S46 peptidase family. The enhancement of four DPP activities was conclusively demonstrated in P. endodontalis, and remarkable Lys-Ala-MCA-hydrolysis was achieved by qualitative and quantitative potentiation of the DPP5 molecule.

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Section: Discussionmentioning

confidence: 99%

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

et al. 2014

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“…The N-terminal Ser of the active SprE was numbered as the first amino acid residue (Ser1). In vitro mutagenesis was performed as reported previously [30] by PCR with mutated primer(s) to substitute 3 amino acids in the prosequence (Glu -15 Ser, Glu -14 Lys, Glu -8 Ile, designated as SprE-mut), 4 amino acids in the mature region (Glu11Gln, Glu6Gln, Ser1Thr/Ala/Val and Leu2Val), and an essential Ser 180 to Ala. All mutations were confirmed by DNA sequencing.…”

Section: Amino Acid Numbering and In Vitro Mutagenesismentioning

confidence: 99%

“…The autodegradation of Staphylococcal glutamyl endopeptidases occurring within the proseuqence region was efficiently suppressed by the substitution of Glu and Asp in the proseqquences, to Gln, Asn or other amino acids [25,30]. Here, this strategy was introduced at the N-terminal region of mature SprE.…”

Section: Suppression Of Auto-degradation Of Mature Sprementioning

confidence: 99%

In Vitro Processing of Glutamyl Endopeptidase Proenzymes from Enterococcus faecalis and Importance of N-terminal Residue in Enzyme Catalysis

Rouf

Ohara‐Nemoto

Ono

et al. 2013

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Glutamyl endopeptidase from Enterococcus faecalis, designated SprE, is one of the important virulence factors secreted as zymogen. In the present study we expressed recombinant SprE proenzyme (pro-SprE) in Escherichia coli and investigated the in vitro processing to mature SprE. It was found that trypsin could efficiently produce the active form of SprE with the N-terminus Ser 1 through cleavage between Arg-1 and Ser 1 bond, which was subsequently auto-degraded into inactive species through the cleavage at the Glu 6-Asp 7 and Glu 11-Val 12 bonds. Although thermolysin could produce SprE with the N-terminus Leu 2 , but possessed no proteolytic activity. In contrast to the absolute requirement of the N-terminal Val 1 in staphylococcal glutamyl endopeptidases, the N-terminal Ser 1 of mature SprE could be substituted by other amino acids despite that Ser showed the maximal activity. Substitution of penultimate Leu 2 of SprE to Val 2 also reduced the activity to 40% of the wild type. Taken together, we conclude that pro-SprE was converted to mature form with the N-terminus Ser 1 by a protease with specificity of trypsin and the length of the N-terminal region rather than specific residue is absolutely required for enzyme activity.

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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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An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

Cited by 6 publications

References 20 publications

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

Identification of Dipeptidyl-Peptidase (DPP)5 and DPP7 in Porphyromonas endodontalis, Distinct from Those in Porphyromonas gingivalis

In Vitro Processing of Glutamyl Endopeptidase Proenzymes from Enterococcus faecalis and Importance of N-terminal Residue in Enzyme Catalysis

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

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