TY - JOUR
T1 - Functional characterization of the Mycobacterium tuberculosis zinc metallopeptidase Zmp1 and identification of potential substrates
AU - Petrera, Agnese
AU - Amstutz, Beat
AU - Gioia, Magda
AU - Hähnlein, Janine
AU - Baici, Antonio
AU - Selchow, Petra
AU - Ferraris, Davide M.
AU - Rizzi, Menico
AU - Sbardella, Diego
AU - Marini, Stefano
AU - Coletta, Massimo
AU - Sander, Peter
N1 - Funding Information:
We acknowledge the support from the Functional Genomics Center Zurich (FGCZ). This work was also supported in part by the Swiss National Science Foundation (3100A0-135705 to P.S.), the Italian Ministry for University and Research (PRIN 200993WWF9 to M.C.), and the European Union (EU-PF7 New TB Vac, Project No. 241745 and EU-PF7 SysteMtb Collaborative Project no. 241587). B.A. was supported by the Forschungskredit 2010 from University of Zurich (54232101).
PY - 2012/7
Y1 - 2012/7
N2 - 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, neuro-tensin, 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 spec-trometry, 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.
AB - 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, neuro-tensin, 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 spec-trometry, 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.
KW - Drug development
KW - Enzyme kinetics
KW - Metallo-peptidase
KW - Mycobacterium tuberculosis
KW - Peptide hormones
KW - Phagosome maturation
UR - http://www.scopus.com/inward/record.url?scp=84867827439&partnerID=8YFLogxK
U2 - 10.1515/hsz-2012-0106
DO - 10.1515/hsz-2012-0106
M3 - Article
SN - 1431-6730
VL - 393
SP - 631
EP - 640
JO - Biological Chemistry
JF - Biological Chemistry
IS - 7
ER -