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Substrate specificity of MarP, a periplasmic protease required for resistance to acid and oxidative stress in Mycobacterium tuberculosis.

TitleSubstrate specificity of MarP, a periplasmic protease required for resistance to acid and oxidative stress in Mycobacterium tuberculosis.
Publication TypeJournal Article
Year of Publication2013
AuthorsSmall JL, O'Donoghue AJ, Boritsch EC, Tsodikov OV, Knudsen GM, Vandal O, Craik CS, Ehrt S
JournalJ Biol Chem
Volume288
Issue18
Pagination12489-99
Date Published2013 May 03
ISSN1083-351X
KeywordsHydrogen-Ion Concentration, Mutation, Mycobacterium tuberculosis, Oxidative Stress, Peptide Hydrolases, Periplasmic Proteins, Protein Folding, Protein Structure, Secondary, Substrate Specificity
Abstract

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.

DOI10.1074/jbc.M113.456541
Alternate JournalJ Biol Chem
PubMed ID23504313
PubMed Central IDPMC3642297
Grant ListP41 GM103481 / GM / NIGMS NIH HHS / United States
8P41GM103481 / GM / NIGMS NIH HHS / United States
AI08172 / AI / NIAID NIH HHS / United States
P50 GM082250 / GM / NIGMS NIH HHS / United States
T32 GM007739 / GM / NIGMS NIH HHS / United States

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