Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M

Yokoi, Kohei; Kakikawa, Makiko; Kimoto, Hisashi; Watanabe, Kouichi; Yasukawa, Hiroo; Yamakawa, Ayanori; Taketo, Akira; Kodaira, Ken-Ichi

doi:10.1016/s0378-1119(01)00782-x

Cited by 35 publications

(58 citation statements)

References 16 publications

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“…AJ, Staphylococcus sp. strain AJ (this study); V8, from S. epidermidis ATCC 12228 [23]; glutamic, glutamic acidspeciWc endoprotease from S. aureus ATCC 12600 [24]; glutamyl, glutamyl endopeptidase from S. warneri M [25]. Underline indicates the N-terminal amino acid position of 11 kDa fragment of AJ Fig.…”

mentioning

confidence: 88%

“…The N-terminal amino acid sequence of the puriWed enzyme up to residue 17 was Val-Ile-Leu-Pro-Asn-Asn-XArg-His-Gln-Ile-Phe-X-Thr-Thr-Gln-Gly-, and shared a high degree of similarity with the N-terminal sequences of a glutamyl endopeptidase (also known as V8 serine protease) from S. epidermidis ATCC 12228 [23], S. aureus ATCC 12600 [24], and S. warneri M [25].…”

Section: N-terminal Amino Acid Sequence Of the Puriwed Enzymementioning

confidence: 99%

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Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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A novel Wbrinolytic enzyme (AJ) was puriWed from Staphylococcus sp. strain AJ screened from Korean salt-fermented Anchovy-jeot. Relative molecular weight of AJ was determined as 26 kDa by using SDS-PAGE and Wbrin zymography. Based on a 2D gel, AJ was found to consist of three active isoforms (pI 5.5-6.0) with the same N-terminal amino acid sequence. AJ exhibited optimum pH and temperature at 2.5-3.0 and 85°C, respectively. AJ kept 85% of the initial activity after heating at 100°C for 20 min on the zymogram gel. The Michaelis constant (K m ) and K cat values of AJ towards -casein were 0.38 mM and 19.73 s ¡1 , respectively. AJ cleaved the A -chain of Wbrinogen but did not aVect the B -and -chains, indicating that it is an -Wbrinogenase. The Wbrinolytic activity was inhibited by diisopropyl Xuorophosphate, indicating AJ is a serine protease. Interestingly, AJ was very stable at acidic condition, SDS, and heat (100°C), whereas it was easily degraded at neutral and alkaline conditions. In particular, AJ formed an active homo-dimer in the pH range from 7.0 to 8.0. To our knowledge, a similar combination of acid and heat stability has not yet been reported for other Wbrinolytic enzymes.

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mentioning

confidence: 88%

Section: N-terminal Amino Acid Sequence Of the Puriwed Enzymementioning

confidence: 99%

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Choi

Song

Chung

et al. 2008

J Ind Microbiol Biotechnol

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“…The N-terminal sequence of native GluSW was determined to Arg 64 -Ala-Asn-Val 67 , and this form is known to exert caseinolytic and glutamic acid-specific hydrolytic activities [10]. However, the present study clearly demonstrated that GluSW starting at Arg 64 had no proteolytic activity and that the hydrolysis of the Asn 66 -Val 67 bond was prerequisite for the maturation.…”

Section: Discussionmentioning

confidence: 50%

“…In contrast, the 31-kDa mature form of GluSW was reported to retain Arg 64 -Ala-Asn 66 at its N-terminus [10], which is equivalent to the C-terminal tripeptide, His 66 -Ala-Asn 68 , of the GluV8 prosequence (Fig. 1a).…”

Section: Identification Of the N-terminus Of Mature Gluswmentioning

confidence: 97%

“…GluSE does not have the C-terminal repeat sequence found in GluV8 [9]. Another Staphylococcal homolog, glutamyl endopeptidase from Staphylococcus warneri (designated GluSW), was cloned and deduced to be composed of 316 amino acids and to possess a 34-amino acid Asp/Asn-rich region at its C-terminus [10]. When GluV8 with its C-terminal repeat deleted was expressed in E. coli, it could fold to an active form after denaturation-renaturation 5 treatment [11].…”

Section: Introductionmentioning

confidence: 99%

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

Ono

Nemoto

Shimoyama

et al. 2008

Analytical Biochemistry

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V8 protease (GluV8), a member of the glutamyl endopeptidase I family, isolated from the V8 strain of Staphylococcus aureus, is widely utilized for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the auto-proteolysis: the use of the prosequence of its homolog (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of 4 substitutions in the prosequence of GluV8. In the present study, we refined this

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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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Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M

Cited by 35 publications

References 16 publications

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

Purification and characterization of a novel thermoacid-stable fibrinolytic enzyme from Staphylococcus sp. strain AJ isolated from Korean salt-fermented Anchovy-joet

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

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

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