Characterization of two M17 family members in Escherichia coli, Peptidase A and Peptidase B.
Escherichia coli encodes two aminopeptidases belonging to the M17 family: Peptidase A (PepA) and Peptidase B (PepB). To gain insights into their substrate specificities, PepA or PepB were overexpressed in Delta pepN, which shows greatly reduced activity against the majority of amino acid substrates. Overexpression of PepA or PepB increases catalytic activity of several aminopeptidase substrates and partially rescues growth of Delta pepN during nutritional downshift and high temperature stress. Purified PepA and PepB display broad substrate specificity and Leu, Lys, Met and Gly are preferred substrates. However, distinct differences are observed between these two paralogs: PepA is more stable at high temperature whereas PepB displays broader substrate specificity as it cleaves Asp and insulin B chain peptide. Importantly, this strategy, i.e. overexpression of peptidases in Delta pepN and screening a panel of substrates for cleavage, can be used to rapidly identify peptidases with novel substrate specificities encoded in genomes of different organisms.
Bhosale
Manoj
M
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Pande
Samay
S
Kumar
Anujith
A
Kairamkonda
Subhash
S
Nandi
Dipankar
D
D000626:Aminopeptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D006868:Hydrolysis; D013379:Substrate Specificity
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C496211:PepA protein, E coli; C075633:pepN protein, Bacteria; D000626:Aminopeptidases; C026045:tripeptide aminopeptidase; D004152:Dipeptidyl-Peptidases and Tripeptidyl-Peptidases