Characterization of a novel intracellular endopeptidase of the alpha/beta hydrolase family from Streptomyces coelicolor A3(2).
In a proteasome-lacking mutant of Streptomyces coelicolor A3(2), an intracellular enzyme with chymotrypsin-like 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 alpha/beta 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.
Nagy
István
I
Centre of Microbial and Plant Genetics, Catholic University of Leuven, Belgium.
Banerjee
Tisha
T
Tamura
Tomohiro
T
Schoofs
Geert
G
Gils
Ann
A
Proost
Paul
P
Tamura
Noriko
N
Baumeister
Wolfgang
W
De Mot
René
R
D000595:Amino Acid Sequence; D003546:Cysteine Endopeptidases / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D017353:Gene Deletion; D006867:Hydrolases / Q000145:classification*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D006867:Hydrolases; D012697:Serine Endopeptidases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex