Characterization of a Novel Intracellular Endopeptidase of the α/β Hydrolase Family from Streptomyces coelicolor A3(2)

Abstract

In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsinlike activity, absent from the wild type, was detected. Complementation that restored proteasome function did not suppress expression of the endopeptidase. Since the enzyme was not found in two other S. coelicolor proteasome mutants, its expression probably resulted from a secondary mutation arisen in the proteasome mutant. Purification of the endopeptidase revealed its identity to SCO7095, a putative hydrolase encoded by the S. coelicolor A3(2) genome with no known homologue. Based on the prediction of a Ser-Asp-His catalytic triad and an ␣/␤ hydrolase fold, SCO7095 was assigned to peptidase clan SC. N-terminally His-tagged SCO7095 was efficiently expressed in Escherichia coli cells and purified for further characterization. Although SCO7095 is distantly related to several proline iminopeptidases, including Thermoplasma acidophilum tricorn-interacting F1, no aminopeptidase activity was detected. On synthetic substrates, the monomeric enzyme exhibited not only chymotrypsin-like activity but also thrombin-like activity.

The Mycobacterium tuberculosis Proteasome Active Site Threonine Is Essential for Persistence Yet Dispensable for Replication and Resistance to Nitric Oxide

“…T. acidophilum proteasome mutants were impaired for survival post heat shock but not under normal growth conditions [38] . Deletion of the proteasome in S. lividans , S. coelicolor and M. smegmatis did not reveal phenotypic defects [39] , [40] , [41] . The current work proves that the 20S proteasome is not essential for growth of M. tuberculosis .…”

Identification and partial characterization of a novel serpin fromEudiplozoon nipponicum(Monogenea, Polyopisthocotylea)

“…In order to detect any possible residual contamination with inhibitory effect in the sample of purified rEnSerp1 expressed in bacteria, a control with bacterial lysate instead of rEnSerp1 was tested. In this case, enhanced hydrolytic activity towards the substrate was observed, which might reflect the presence of active bacterial peptidases in the sample that would cleave the GPR [68]. Nevertheless, the bacterial lysate sample alone did not show any inhibitory effect, which indicates that the inhibition achieved in our measurements was related solely to rEnSerp1.…”

The N-terminal coiled coil of the Rhodococcus erythropolis ARC AAA ATPase is neither necessary for oligomerization nor nucleotide hydrolysis

“…In contrast, in Bacteria the occurrence of 20S proteasomes is restricted to the class Actinobacteria (De Mot et al, 1999). The proteasome was found to be nonessential based on two knock-out studies performed in the actinobacteria Mycobacterium smegmatis (Knipfer and Shrader, 1997) and Streptomyces coelicolor (Nagy et al, 2003). A recent analysis of sequenced genomes revealed that not all members of the Actinobacteria contain 20S proteasome genes (this paper).…”