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Cysteine sulfonic acid (Cys-SO3H; cysteic acid) is an oxidative post-translational modification of cysteine, resulting from further oxidation from cysteine sulfinic acid (Cys- SO2H). Cysteine sulfonic acid is considered an irreversible post-translational modification, which serves as a biomarker of oxidative stress that has resulted in oxidative damage to proteins. Cysteine sulfonic acid is anionic, as a sulfonate (Cys-SO –; cysteate), in the ionization state that 3 is almost exclusively present at physiological pH (pKa ~ –2). In order to understand protein structural changes that can occur upon oxidation to cysteine sulfonic acid, we analyzed its conformational preferences, using experimental methods, bioinformatics, and DFT-based computational analysis. Cysteine sulfonic acid was incorporated into model peptides for α-helix and polyproline II helix (PPII). Within peptides, oxidation of cysteine to the sulfonic acid proceeds rapidly and efficiently at room temperature in solution with methyltrioxorhenium (MeReO3) and H2O2. Peptides containing cysteine sulfonic acid were also generated on solid phase using trityl-protected cysteine and oxidation with MeReO3 and H2O2. Using methoxybenzyl (Mob)-protected cysteine, solid-phase oxidation with MeReO3 and H2O2 generated the Mob sulfone precursor to Cys-SO – within fully synthesized peptides. These two solid-phase methods allow the synthesis of peptides containing either Cys-SO – or Cys-SO – in a 32 practical manner, with no solution-phase synthesis required. Cys-SO – had low PPII propensity 3 for PPII propagation, despite promoting a relatively compact conformation in φ. In contrast, in a PPII initiation model system, Cys-SO – promoted PPII relative to neutral Cys, with PPII initiation similar to Cys thiolate but less than Cys-SO – or Ala. In an α-helix model system, Cys- 2 SO – promoted α-helix near the N-terminus, due to favorable helix dipole interactions and 3 favorable α-helix capping via a sulfonate-amide side chain-main chain hydrogen bond. Across all peptides, the sulfonate side chain was significantly less ordered than that of the sulfinate. Analysis of Cys-SO – in the PDB revealed a very strong propensity for local (i/i or i/i+1) side 3 chain-main chain sulfonate-amide hydrogen bonds for Cys-SO –, with > 80% of Cys-SO – 33 residues exhibiting these interactions. DFT calculations conducted to explore these conformational preferences indicated that side chain-main chain hydrogen bonds of the sulfonate with the intraresidue amide and/or with the i+1 amide were favorable. However, hydrogen bonds to water or to amides, as well as interactions with oxophilic metals, were weaker for the sulfonate than the sulfinate, due to lower charge density on the oxygens in the sulfonate.
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This content will become publicly available on August 1, 2026
Biochemical Consequences of a Leucine-to-Cysteine Clamp Substitution in Lipoxygenases
Lipoxygenases (LOXs) are a family of metalloenzymes that oxidize polyunsaturated fatty acids producing cell-signaling hydroperoxides. Fungal LOXs have drawn interest because of their roles in plant and animal pathogenesis. A new subfamily of annotated fungal LOXs has been predicted. One of its unique structural features is the presence of a cysteine amino acid encoded at the invariant leucine clamp. Herein, we isolate three representatives of this LOX subfamily from recombinant expressions in both yeast and bacterial cultures. Metal analysis indicates that the proteins accommodate a mononuclear manganese ion center, similar to other eukaryotic LOXs, but have nominal LOX activity. The functional consequence of the non-conservative mutation is further explored using a Leu-to-Cys (L546C) variant of soybean lipoxygenase, a model plant orthologue. While this L546C variant has comparable structural integrity and metal content to the native enzyme, the variant is associated with a 50-fold decrease in the first-order rate constant. The presence of cysteine at 546, compared to leucine, alanine, or serine, also results in a distinctive kinetic lag phase and product inhibition. The collective data highlight that Cys encoded at the Leu clamp is detrimental to LOX activity. Potential biological functions of these annotated fungal LOXs are discussed.
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- Award ID(s):
- 2231079
- PAR ID:
- 10657471
- Publisher / Repository:
- MDPI
- Date Published:
- Journal Name:
- Biomolecules
- Volume:
- 15
- Issue:
- 8
- ISSN:
- 2218-273X
- Page Range / eLocation ID:
- 1153
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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