title: S-nitrosylation of Mycobacterium tuberculosis tyrosine phosphatase A (PtpA) induces its structural instability.
abstract: S-nitrosylation is associated with signal transduction and microbicidal activity of nitric oxide (NO). We have recently described the S-nitrosylation of Mycobacterium tuberculosis protein tyrosine phosphatase A, PtpA, an enzyme that plays an important role in mycobacteria survival inside macrophages. This post-translational modification decreases the activity of the enzyme upon modification of a single Cys residue, C53. The aim of the present work was the investigation of the effect of S-nitrosylation in PtpA kinetic parameters, thermal stability and structure. It was observed that the K(M) of nitrosylated PtpA was similar to its unmodified form, but the V(max) was significantly reduced. In contrast, treatment of PtpA C53A with GSNO, did not alter either K(M) or V(max). These results confirmed that PtpA S-nitrosylation occurs specifically in the non-catalytic C53 and that this modification does not affect substrate affinity. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy techniques it was shown that PtpA S-nitrosylation decreased protein thermal stability and promoted a local effect in the surroundings of the C53 residue, which interfered in both protein stability and function.
authors.affiliation: Centro de Biologia Molecular Estrutural-INBEB, Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianópolis, Brazil.
authors.forename: Camila
authors.initials: C
authors.lastname: Matiollo
authors.forename: Gabriela
authors.initials: G
authors.lastname: Ecco
authors.forename: Angela Camila Orbem
authors.initials: AC
authors.lastname: Menegatti
authors.forename: Guilherme
authors.initials: G
authors.lastname: Razzera
authors.forename: Javier
authors.initials: J
authors.lastname: Vernal
authors.forename: Hernán
authors.initials: H
authors.lastname: Terenzi
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004795:Enzyme Stability / Q000235:genetics; D006801:Humans; D008264:Macrophages / Q000201:enzymology / Q000378:metabolism; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics; D009569:Nitric Oxide / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D017434:Protein Structure, Tertiary; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C414465:MptpA protein, Mycobacterium tuberculosis; D009569:Nitric Oxide; D017027:Protein Tyrosine Phosphatases