Inhibition of Arabidopsis O-acetylserine(thiol)lyase A1 by tyrosine nitration

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.

FIGURE 1.

Effect of nitration on OASTL activity and OASA1 protein levels of wild type and mutant extracts. Total OASTL activity and OASA1 protein levels were analyzed in wild type and oasa1.1 extracts obtained either in the presence (+DTT) or absence of (−DTT), and either non-nitrated as control (C) or nitrated (N) by treatment with 1 mm nitrite/hydrogen peroxide mixture for 1 h. Anti-OASA1 antibody crossreacted preferentially with OASA1 but showed also a weaker crossreaction with OASB isoform. Ponceau S staining of Rubisco is shown as loading control. OASTL activities are the mean values of three replicates ± S.D. (A). OASTL activity in 0.5 mm SIN-1 treated extracts from wild type, oasa1.1, oas1.2, oasb, and oasc mutants was measured after 1 h. After SIN-1 removal, activity was measured along with non-treated samples as controls. Values are the mean ± S.D. of three replicates and are expressed relative to the corresponding non-nitrated control for every genotype (B).

FIGURE 2.

OASTL activity and nitrated OASA1 protein levels in purified OASA1 protein treated with increasing concentrations of the nitrating reagent SIN-1. Samples of 12 μg of His-tagged purified recombinant OASA1 protein were treated with the indicated concentration of SIN-1 for 1 h. After SIN-1 removal, OASTL activity, and total and nitrated protein levels were quantified (A). Activity and protein levels in samples treated with 0.5 mm SIN-1 or untreated as a control (c) and epicatechin at the indicated concentrations (B). Western blot with anti-3nitroY antibody (α-3NY) and Ponceau staining as loading control are shown in panels A and B. Scavenging effect of epicatechin on the OASTL activity of SIN-1 (0.5 mm)-nitrated crude extracts from wild type (clear gray bars) and oasa1.1 (dark gray bars) leaves (C). OASTL activities are the mean values of three replicates ± S.D.

FIGURE 3.

Identification of a Tyr-nitration site in OASA1. LC-MS/MS analysis of nitrated OASA1 and non-nitrated control allowed the identification of Y*LSTVLFDATR peptide with nitrated or non-nitrated Tyr³⁰², respectively (A). The 22.49 Da shift of the double charged peptide indicates nitration of Tyr³⁰². Ion score (MASCOT) of the nitrated and non-nitrated peptides are shown. The MS spectra corresponding to Y(NO₂)LSTVLFDATR (Z = 2; m/z = 665.82) is shown (B). NO₂-Y indicates the presence of the nitrated tyrosine immonium ion mass in the LC-MS/MS spectra. Identified y and b ion series are pointed out.

FIGURE 4.

Three-dimensional model of OASA1 showing the position and potential interactions of Tyr residues. The conformation of OASA1 molecule showing the position of the seven Tyr residues (in red), the PLP binding site (in orange), O-acetylserine substrate binding site (in yellow) and SAT protein interaction site (in purple) are shown (A). Detail of the three-dimensional structure showing Tyr³⁰² and the Asn⁷⁷ residues at a 4.7 Å distance, the amino acid residues interacting with the Lys⁴⁶ (in orange)-linked PLP (in yellow) through hydrogen bonds (in green), and the OAS and SAT binding site (in purple) (B). In silico analysis, using PyMol software, of the potential hydrogen bonds (yellow dashed line) between amino acid residues surrounding PLP (C). Spectrophotometric detection of the reaction of OASA1-bound O-acetylserine with PLP to form an α-aminoacrylate intermediate. Samples of 200 μg of non-nitrated (open circles) or nitrated (close circles) purified OASA1 were incubated with increasing concentrations of O-acetylserine as indicated, and the increase in the absorbance at 470 nm registered (D).

FIGURE 5.

OASTL activity of mutated recombinant versions of OASA1. Purified samples of WT and mutated (Y302A and Y203A) versions of OASA1 were assayed for OASTL activity in the absence or presence of the indicated concentrations of SIN-1. OASTL activities are the mean values of three replicates ± standard deviation. Western blot with anti-3nitroY antibody (α-3NY) and Ponceau staining as loading control (Ponceau) are shown for nitrated Y203A samples.

FIGURE 6.

Analysis of in vivo Tyr-nitration of OASA1. Crude extracts from wild-type A. thaliana seedlings were immunoprecipitated with anti-3-nitroY antibody, and the presence of OASA1 in crude extract (CE) input and the resulting supernatant (Sup) and immunoprecipitate (IP) was analyzed by Western blot with anti-OASA1 antibody (A). Leaves were infiltrated with 2 mm SIN-1 for 90 min and then crude protein extracts were prepared for OASTL activity measurement (bottom panel) and for immunoprecipitation with 3-nitroTyr (IP 3-NY) antibodies. Western blot with anti-OASA1 (α-OASA1) was performed with protein extracts (left up panel) or IP (right up panel). OASTL activity values are the mean values of three replicates ± standard deviation (B).