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

Abstract

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.

New microbial resource: microbial diversity, function and dynamics in Chinese liquor starter

“…Meanwhile, the highly abundant no-rank Actinobacteria in JF liquor starter (J3 and J4) could produce volatile organic compounds; the secondary metabolic products of Actinomyces could also contribute to flavor development 38 . Thermoactinomyces can produce plenty of thermostable extracellular proteolytic enzymes, e.g., alkaline proteinase 39 , glutamyl endopeptidase 40 and carboxypeptidase 41 . This study further found that Bacillales and no-rank Actinobacteria were the dominant communities when the temperature increased to the highest point.…”

Complete Genome Sequence of Thermoactinomyces vulgaris Strain CDF, a Thermophilic Bacterium Capable of Degrading Chicken Feathers

“…The colonies were picked and restreaked until the strain was axenic, and the pure culture was deposited in the China Center for Type Culture Collection (CCTCC) under the accession number AB206328. Strain CDF has the ability to degrade chicken feathers, and three proteases of this bacterium have been previously characterized, including a spore-associated protease (6), a subtilisin-like keratinolytic protease (7), and a glutamyl endopeptidase (8).…”

Glutamyl Endopeptidases: The Puzzle of Substrate Specificity

“…ET precursors contain only a secretory leader [3] and, therefore, are processed by signal peptidase. For bacterial GEPases similar to Glu-V8 and BIGEP, propeptide is removed heterocatalytically by different proteases [22, 100-104], with just one exception [21]. Comparison of the structures of the S1 sites of GEPases and the processing mechanisms shows that no explicit substrate charge compensator is revealed in the S1 pocket in autoactivated enzymes; the S1 site of ET is characterized by the presence of the Lys216 residue, while the GEPases similar to Glu–V8 and BIGEP processed heterocatalytically contain an α-amino group of the N-terminal residue.…”

“…Meanwhile, data have been published demonstrating that the precursors of Glu-V8 [113], BIGEP [109], Esp [7], as well as GEPases from B. licheniformis [114], B. subtilis [102], and Thermoactinomyces sp. [21] are capable of autoprocessing; in most cases, it is the bonds corresponding to the specificity of the mature enzymes that are cleaved [7, 21, 109, 114]. Furthermore, glutamate activity in trans of precursor analogues has been detected [100].…”

“…To make the picture complete, we would like to add that data on the enzyme from Thermoactinomyces sp. carrying Glu1, which can be autoactivated in vitro in a heterologous expression system [21], identically to the previously artificially obtained mutants of other GEPases [109, 114], have been published recently. Hence, maturation of enzymes similar to Glu–V8 is a stepwise process.…”