In vitro maturation pathway of a glutamyl endopeptidase precursor from Bacillus licheniformis

Expression, purification, and characterization of soluble and active glutamate-specific endopeptidase in Bacillus licheniformis and Pichia pastoris

“…This high enzymatic activity may be attributed to N-glycosylation, which could facilitate the formation of active conformation of full-length GSE-BL and substrate-binding pocket of "His47-Asp96-Ser167" (Fig. 6) [16,22], thus facilitating the binding of GSE-BL-P to substrate Z-Phe-Leu-Glu-pNA. Glycosylation could modify the structure of bovine pancreatic ribonuclease B, and the stability and functional activity of ribonuclease B…”

Glutamyl Endopeptidases: The Puzzle of Substrate Specificity

“…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].…”

“…long rather than in complete deletion of the prosequence [7, 100, 109, 113, 114] that corresponds to the size of propeptides in mammalian CLPs. These species exhibit no activity with respect to protein substrates and low activity with respect to peptides in trans and can be activated only heterocatalytically [7, 100, 109, 113, 114]. To make the picture complete, we would like to add that data on the enzyme from Thermoactinomyces sp.…”

“…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.…”

“…The first one is a long folding assistant [99, 109] that ensures the kinetic stability of a mature protein as often occurs in bacterial proteases [115]. The second propeptide, which is short and forms after the first processing step, is the activation unit [109, 113, 114] that maintains the inactive state of the enzyme. Furthermore, it cannot be ruled out that the structure of the active site of proteases changes after the first propeptide portion is removed.…”