Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity

Technical Summary: Amino Acids in the RSSY Motif of Lipoyl Synthase Control Substrate Binding and Reactivity

Problem Statement
Lipoyl synthase (LipA) catalyzes the final step in the biosynthesis of the lipoyl cofactor, inserting two sulfur atoms at the C6 and C8 positions of an octanoyl chain attached to a lipoyl carrier protein. This reaction relies on the degradation of an auxiliary [4Fe-4S] cluster to supply sulfur atoms. While the general mechanism involving radical S-adenosylmethionine (SAM) chemistry and cluster disassembly is established, the specific structural roles of the highly conserved C-terminal RSSY motif (Arg-Ser-Ser-Tyr) in substrate binding, cluster integrity, and reaction outcome remain incompletely defined. Specifically, the functional necessity of the arginine and the two serine residues, and how they influence the fate of the auxiliary cluster and the reaction pathway, require detailed biochemical and spectroscopic characterization.

Methodology
The study employed a combination of site-directed mutagenesis, biochemical assays, and advanced spectroscopic techniques using Escherichia coli LipA (Ec LipA).

• Mutagenesis and Activity Assays: Variants of the RSSY motif (R306K, S307A, S308A, S308T, S308F, S308C, Y309F) were generated. Enzymatic activity was assessed using liquid chromatography-mass spectrometry (LC/MS) to quantify the formation of the lipoyl product, the 6-mercaptooctanoyl intermediate, and potential side products. Assays were conducted with both the native octanoyl peptide substrate and a non-native 8-mercaptooctanoyl peptide (8-MOA), with and without the cluster carrier protein Ec NfuA to test for multiple turnover capabilities.
• Binding Studies: Isothermal titration calorimetry (ITC) was used to measure the binding affinity of the octanoyl substrate to wild-type (WT) and R306K LipA. Temperature-Related Intensity Change (TRIC) assays determined SAM binding affinity.
• Spectroscopic Characterization: Mössbauer spectroscopy and Electron Paramagnetic Resonance (EPR) spectroscopy were utilized to monitor the oxidation states and spin states of the iron-sulfur clusters during the reaction, particularly focusing on the S308C variant.
• Product Identification: The desaturated product formed by the S308C variant was purified and characterized using 13C NMR spectroscopy with isotopically labeled substrates ([6,6-2H2], [8,8,8-2H3], and [7,8-13C2] octanoyl peptides) to distinguish between cyclopropyl and alkene structures.

Key Results

1. Role of Arg306 in Substrate Binding: Arg306 is absolutely essential for catalysis. The R306K variant is essentially inactive, failing to form lipoyl product or accumulate significant intermediates. ITC data revealed that Arg306 forms critical hydrogen bonds with the octanoyl carbonyl and hydrophobic contacts with the acyl chain. Its substitution reduces substrate binding affinity by over three orders of magnitude, indicating Arg306 is pivotal for substrate positioning.
2. Essentiality of Ser308 and Divergent Reactivity: Ser308, which ligates the unique iron of the auxiliary [4Fe-4S] cluster, is critical.
   • S308A and S308F: These variants are largely inactive with the native substrate but show unexpected activity with the 8-MOA substrate, particularly in the presence of NfuA, suggesting the removal of the serine ligand alters the cluster's accessibility.
   • S308C: This variant retains the ability to form the 6-mercaptooctanoyl intermediate but fails to produce the lipoyl cofactor. Instead, it accumulates a desaturated product identified as 6-octenoyl-lysyl peptide.
3. Mechanistic Divergence in S308C:
   • Cluster Integrity: Unlike the wild-type enzyme, where the auxiliary cluster degrades to a [3Fe-4S] species during turnover, the S308C variant retains a tetranuclear [4Fe-4S] cluster throughout the reaction.
   • Spectroscopic Evidence: The reaction intermediate in S308C exhibits a high-spin, S = 7/2 ground state, assigned to a monothiolated [4Fe-4S]+ cluster. This species is transient and decays to the 6-octenoyl product.
   • Reaction Pathway: The data suggest that the stronger cysteine ligand in S308C prevents the release of the unique iron atom. Consequently, the second hydrogen abstraction occurs at C7 rather than C8. The resulting radical undergoes recombination that cleaves the C6–S bond, restoring the auxiliary cluster and releasing the desaturated alkene product, rather than proceeding to the second sulfur insertion.
4. Role of Ser307 and Tyr309: Substitutions at these positions (S307A, Y309F) result in reduced but detectable activity, indicating they are not strictly essential for catalysis but likely contribute to structural positioning of the C-terminal tail.

Significance and Claims
The paper establishes that the RSSY motif acts as a critical regulatory switch for LipA function.

• Substrate Recognition: Arg306 is identified as the primary anchor for substrate binding, mediating specific interactions with the octanoyl chain.
• Cluster Dynamics and Reaction Fate: The study demonstrates that the ligation of the unique iron by Ser308 is the key determinant for the reaction outcome. The weak ligation by serine in the wild-type enzyme facilitates the release of the unique iron, a step necessary for the degradation of the auxiliary cluster and the successful insertion of the second sulfur atom.
• Mechanistic Plasticity: The S308C variant reveals that preventing the release of the unique iron (via the stronger cysteine ligand) blocks the canonical sulfur insertion pathway. Instead, it redirects the reaction toward an alternative pathway involving C7 abstraction and C–S bond cleavage, yielding a desaturated product. This highlights that the structural integrity of the auxiliary cluster and the specific ligation environment of the unique iron are decisive factors in determining whether the enzyme performs sulfur insertion or catalyzes desaturation.

The authors conclude that the RSSY motif, particularly the interplay between Arg306 and Ser308, controls both the initial substrate binding and the subsequent mechanistic branching of the radical reaction, ensuring the fidelity of lipoyl cofactor biosynthesis.