In Vitro Processing of Glutamyl Endopeptidase Proenzymes from Enterococcus faecalis and Importance of N-terminal Residue in Enzyme Catalysis
Shakh M. A. Rouf,
Y. Ohara-Nemoto,
T. Ono,
Y. Shimoyama,
S. Kimura,
T. K. Nemoto
Issue:
Volume 1, Issue 5, December 2013
Pages:
73-80
Received:
22 November 2013
Accepted:
Published:
30 December 2013
Abstract: 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 Ser1 through cleavage between Arg-1 and Ser1 bond, which was subsequently auto-degraded into inactive species through the cleavage at the Glu6-Asp7 and Glu11-Val12 bonds. Although thermolysin could produce SprE with the N-terminus Leu2, but possessed no proteolytic activity. In contrast to the absolute requirement of the N-terminal Val1 in staphylococcal glutamyl endopeptidases, the N-terminal Ser1 of mature SprE could be substituted by other amino acids despite that Ser showed the maximal activity. Substitution of penultimate Leu2 of SprE to Val2 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 Ser1 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.
Abstract: 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...
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Endo-N-Acetyl-β-D-Glucosaminidases and Peptide-N4-(N-acetyl-β-D-Glucosaminyl) Asparagine Amidases: More Than Just Tools
Issue:
Volume 1, Issue 5, December 2013
Pages:
81-99
Received:
13 December 2013
Accepted:
Published:
30 December 2013
Abstract: Since the discovery of endo-N-acetyl-β-D-glucosaminidases (ENGase) and peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidases (PNGase) most of the published work described their use for structural studies. Less attention was given to the potential roles of those enzymes in the physiology of the cells/organisms they produced them. The scope of this review is firstly to analyse the data on the occurrence and characteristics of murein-, chitin-, and N-glycan-ENGases acting on GlcNAc-containing polymers in three structural families, namely murein, chitin, and N-glycosylproteins, and of PNGases, only acting on N-glycosylproteins, and secondly to discuss the biological roles of the enzymes in the producing cells. The analysis demonstrates the remarkable diversity of the enzymes, and simultaneously the interest of studying their substrate specificity and their structural features. Many examples illustrate the importance of the structure/function relationships studies. Diverse biological roles were anticipated, e.g. they are useful for feeding purposes, are implicated in pathogenesis processes, modulate the activity of macromolecules, and help in the destruction of misfolded proteins. Their effect can be direct or indirect, through the reaction products. Current knowledge only partially explains the biological roles of ENGases and PNGases, thus further studies are expected for determining novel possibilities and elucidating other cell pathways.
Abstract: Since the discovery of endo-N-acetyl-β-D-glucosaminidases (ENGase) and peptide-N4-(N-acetyl-β-D-glucosaminyl) asparagine amidases (PNGase) most of the published work described their use for structural studies. Less attention was given to the potential roles of those enzymes in the physiology of the cells/organisms they produced them. The scope of t...
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