Browsing by Author "Fuentes-Lemus, Eduardo"

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    M. jannaschii FtsZ, a key protein in bacterial cell division, is inactivated by peroxyl radical-mediated methionine oxidation
    (Elsevier, 2021) Reyes Valenzuela, Juan Sebastian; Fuentes-Lemus, Eduardo; Aspée, Alexis; Davies, Michael J.; Monasterio, Octavio; López Alarcón, Camilo Ignacio
    Oxidation and inactivation of FtsZ is of interest due to the key role of this protein in bacterial cell division. In the present work, we studied peroxyl radical (from AAPH, 2,2′-azobis(2-methylpropionamidine)dihydrochloride) mediated oxidation of the highly stable FtsZ protein (MjFtsZ) from M. jannaschii, a thermophilic microorganism. MjFtsZ contains eleven Met, and single Tyr and Trp residues which would be expected to be susceptible to oxidation. We hypothesized that exposure of MjFtsZ to AAPH-derived radicals would induce Met oxidation, and cross-linking (via di-Tyr and di-Trp formation), with concomitant loss of its functional polymerization and depolymerization (GTPase) activities. Solutions containing MjFtsZ and AAPH (10 or 100 mM) were incubated at 37 °C for 3 h. Polymerization/depolymerization were assessed by light scattering, while changes in mass were analyzed by SDS-PAGE. Amino acid consumption was quantified by HPLC with fluorescence detection, or direct fluorescence (Trp). Oxidation products and modifications at individual Met residues were quantified by UPLC with mass detection. Oxidation inhibited polymerization-depolymerization activity, and yielded low levels of irreversible protein dimers. With 10 mM AAPH only Trp and Met were consumed giving di-alcohols, kynurenine and di-Trp (from Trp) and the sulfoxide (from Met). With 100 mM AAPH low levels of Tyr oxidation (but not di-Tyr formation) were also observed. Correlation with the functional analyses indicates that Met oxidation, and particularly Met164 is the key driver of MjFtsZ inactivation, probably as a result of the position of this residue at the protein-protein interface of longitudinal interactions and in close proximity to the GTP binding site.