Abstract
6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases. Neopterin derived from 7,8-dihydroneopterin triphosphate is secreted by monocytes/macrophages, and is a well-known biomarker for cellular immunity. Because PTS activity in the cell can be a determinant of neopterin production, here we used recombinant human PTS protein to investigate how its activity is regulated, especially depending on redox conditions. Human PTS has two cysteines: Cys-43 at the catalytic site and Cys-10 at the N terminus. PTS can be oxidized and consequently inactivated by H2O2 treatment, oxidized GSH, or S-nitrosoglutathione, and determining the oxidized modifications of PTS induced by each oxidant by MALDI-TOF MS, we show that PTS is S-glutathionylated in the presence of GSH and H2O2S-Glutathionylation at Cys-43 protected PTS from H2O2-induced irreversible sulfinylation and sulfonylation. We also found that PTS expressed in HeLa and THP-1 cells is reversibly modified under oxidative stress conditions. Our findings suggest that PTS activity and S-glutathionylation is regulated by the cellular redox environment and that reversible S-glutathionylation protects PTS against oxidative stress.
Highlights
6-Pyruvoyl tetrahydropterin synthase (PTS) converts 7,8-dihydroneopterin triphosphate into 6-pyruvoyltetrahydropterin and is a critical enzyme for the de novo synthesis of tetrahydrobiopterin, an essential cofactor for aromatic amino acid hydroxylases and nitric-oxide synthases
6-Pyruvoyl tetrahydropterin synthase (PTS)2 catalyzes the second step of the de novo biosynthesis of tetrahydrobiopterin (BH4), an essential cofactor of aromatic amino acid hydroxylases, nitric oxide synthases, and alkylglycerol monooxygenase [1]
We purified recombinant PTS protein using the buffer without DTT as a thiol-protective reagent and found that PTS enzymatic activity was increased by the treatment with DTT as previously reported [25], suggesting that the PTS protein could be subject to oxidative modification
Summary
Tion of inorganic triphosphate followed by oxidation, resulting in neopterin. Neopterin is considered a byproduct of BH4 de novo synthesis [2]. Reversible oxidative modifications and the reduction have an impact on the physiological roles of the proteins and are referred to as a redox regulation (14 –16). Cysteine reversibly binds to GSH (S-glutathionylation) and nitric oxide (S-nitrosylation) and is oxidized mildly by reactive species (sulfenylation) [17,18,19,20,21]. These oxidative modifications can be regenerated by reduced glutathione (GSH), an abundant antioxidant tripeptide that is present at millimolar concentrations in the cell, and reducing proteins such as thioredoxin (Trx) and glutaredoxin (Grx) [22,23,24]. We show that the cysteine residue of PTS is modified in HeLa and THP-1 cells under oxidative stress conditions
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