Substrate Specificity of MarP, a Periplasmic Protease Required for Resistance to Acid and Oxidative Stress in Mycobacterium tuberculosis

Weill Cornell Medical College, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2013; 288(18). DOI: 10.1074/jbc.M113.456541
Source: PubMed


The transmembrane serine protease MarP is important for pH homeostasis in Mycobacterium tuberculosis (Mtb). Previous structural studies revealed that MarP contains a chymotrypsin fold and a disulfide bond that stabilizes the
protease active site in the substrate-bound conformation. Here, we determined that MarP is located in the Mtb periplasm and
showed that this localization is essential for function. Using the recombinant protease domain of MarP, we identified its
substrate specificity using two independent assays: positional-scanning synthetic combinatorial library profiling and multiplex
substrate profiling by mass spectrometry. These methods revealed that MarP prefers bulky residues at P4, tryptophan or leucine
at P2, arginine or hydrophobic residues at P1, and alanine or asparagine at P1′. Guided by these data, we designed fluorogenic
peptide substrates and characterized the kinetic properties of MarP. Finally, we tested the impact of mutating MarP cysteine
residues on the peptidolytic activity of recombinant MarP and its ability to complement phenotypes of Mtb ΔMarP. Taken together,
our studies provide insight into the enzymatic properties of MarP, its substrate preference, and the importance of its transmembrane
helices and disulfide bond.

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Available from: Anthony O'Donoghue, Jan 20, 2016
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