title: Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates.
abstract: Zinc metallopeptidases of bacterial pathogens are widely distributed virulence factors and represent promising pharmacological targets. In this work, we have characterized Zmp1, a zinc metallopeptidase identified as a virulence factor of Mycobacterium tuberculosis and belonging to the neprilysin (NEP; M13) family, whose X-ray structure has been recently solved. Interestingly, this enzyme shows an optimum activity toward a fluorogenic substrate at moderately acidic pH values (i.e., 6.3), which corresponds to those reported for the Mtb phagosome where this enzyme should exert its pathological activity. Substrate specificity of Zmp1 was investigated by screening a peptide library. Several sequences derived from biologically relevant proteins were identified as possible substrates, including the neuropeptides bradykinin, neurotensin, and neuropeptide FF. Further, subsequences of other small bioactive peptides were found among most frequently cleaved sites, e.g., apelin-13 and substance P. We determined the specific cleavage site within neuropeptides by mass spectrometry, observing that hydrophobic amino acids, mainly phenylalanine and isoleucine, are overrepresented at position P1'. In addition, the enzymatic mechanism of Zmp1 toward these neuropeptides has been characterized, displaying some differences with respect to the synthetic fluorogenic substrate and indicating that the enzyme adapts its enzymatic action to different substrates.
authors.affiliation: Institute of Medical Microbiology, University of Zurich, Gloriastrasse, Zurich, Switzerland.
authors.forename: Agnese
authors.initials: A
authors.lastname: Petrera
authors.forename: Beat
authors.initials: B
authors.lastname: Amstutz
authors.forename: Magda
authors.initials: M
authors.lastname: Gioia
authors.forename: Janine
authors.initials: J
authors.lastname: Hähnlein
authors.forename: Antonio
authors.initials: A
authors.lastname: Baici
authors.forename: Petra
authors.initials: P
authors.lastname: Selchow
authors.forename: Davide M
authors.initials: DM
authors.lastname: Ferraris
authors.forename: Menico
authors.initials: M
authors.lastname: Rizzi
authors.forename: Diego
authors.initials: D
authors.lastname: Sbardella
authors.forename: Stefano
authors.initials: S
authors.lastname: Marini
authors.forename: Massimo
authors.initials: M
authors.lastname: Coletta
authors.forename: Peter
authors.initials: P
authors.lastname: Sander
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D006877:Hydroxamic Acids; D007211:Indoles / Q000494:pharmacology; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism; D007700:Kinetics; D045726:Metalloproteases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009479:Neuropeptides / Q000737:chemistry / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D059748:Proteolysis; D013373:Substance P / Q000378:metabolism; D013379:Substrate Specificity; D015032:Zinc / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D006877:Hydroxamic Acids; D007211:Indoles; D036341:Intercellular Signaling Peptides and Proteins; D009479:Neuropeptides; D011480:Protease Inhibitors; C495199:apelin-13 peptide; D013373:Substance P; D045726:Metalloproteases; C568073:Zmp1 protein, Mycobacterium tuberculosis; C119203:ilomastat; D015032:Zinc