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Abstract
The last step of sulfur assimilation is catalyzed by O-acetylserine(thiol)lyase (OASTL) enzymes. OASTLs are encoded by a multigene family in the model plant Arabidopsis thaliana. Cytosolic OASA1 enzyme is the main source of OASTL activity and thus crucial for cysteine homeostasis. We found that nitrating conditions after exposure to peroxynitrite strongly inhibited OASTL activity. Among OASTLs, OASA1 was markedly sensitive to nitration as demonstrated by the comparative analysis of OASTL activity in nitrated crude protein extracts from wild type and different oastl mutants. Furthermore, nitration assays on purified recombinant OASA1 protein led to 90% reduction of the activity due to inhibition of the enzyme, as no degradation of the protein occurred under these conditions. The reduced activity was due to nitration of the protein because selective scavenging of peroxynitrite with epicatechin impaired OASA1 nitration and the concomitant inhibition of OASTL activity. Inhibition of OASA1 activity upon nitration correlated with the identification of a modified OASA1 protein containing 3-nitroTyr(302) residue. The essential role of the Tyr(302) residue for the catalytic activity was further demonstrated by the loss of OASTL activity of a Y302A-mutated version of OASA1. Inhibition caused by Tyr(302) nitration on OASA1 activity seems to be due to a drastically reduced O-acetylserine substrate binding to the nitrated protein, and also to reduced stabilization of the pyridoxal-5'-phosphate cofactor through hydrogen bonds. This is the first report identifying a Tyr nitration site of a plant protein with functional effect and the first post-translational modification identified in OASA1 enzyme.
Highlights
Sulfur is an essential nutrient for all living organisms as it is a component of the amino acids cysteine and methionine required for protein synthesis
Because cysteine biosynthesis requires the reduced sulfur in form of sulfide, which is exclusively produced through sulfate assimilatory pathway in plastids [14], the mitochondria provide the bulk of OAS [2], and the main site for Cys production is cytosol in Arabidopsis, an exchange of sulfide and OAS between subcellular compartments must be important in controlling the function of the enzyme components of the cysteine synthase complex
Cysteine biosynthesis is a crucial process because this amino acid is a constituent of proteins
Summary
Plant Growth Conditions and Treatments—A. thaliana seeds of Col-0 wild type or different oastl mutants [9] were sown in moistened soil and grown under photoperiodic conditions (cycles of 16 h day and 8 h night for long days, at 22 °C and 20 °C). Protein Extraction, Immunoprecipitation, and Nitrating Treatments—For activity assays, around 100 mg of Col-0 and oastl mutant leaves were frozen and ground in liquid nitrogen and extracted by adding extraction buffer (50 mM phosphate buffer, pH 7.5, 1 mM EDTA, 10 M PLP, 0.5 mM PMSF, 1% (v/v) protease inhibitor mixture from Sigma) with or without 1 mM DTT, as described in each case, and briefly vortexing. Nitrating treatments on crude extracts or purified recombinant protein was performed by treatment with peroxynitrite generated from sodium nitrite plus hydrogen peroxide, or 3-morpholinosydnonimine hydrochloride (SIN-1; Invitrogen) at the indicated concentrations, as previously reported [24, 25]. Western Blots—Protein extracts were separated by 10% SDS-PAGE, blotted onto nitrocellulose membrane, stained with Ponceau-S and probed with antibodies at the followed dilutions: monoclonal anti-3-NY (Cayman Chemicals) 1:1000, anti-His (Santa Cruz Biotechnology) 1:500, and custom-made polyclonal anti-recombinant OAS-A1 antibody (Biomedal S.L.) 1:10000. The distance between residues in Angstroms (Å) and the presence of hydrogen bonds were carried out with both programs with default settings
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