LigU from Novosphingobium sp. strain KA1 catalyzes the isomerization of (4E)-oxalomesaconate (OMA) to (3Z)-2-keto-4-carboxy-3-hexenedioate (KCH) as part of the protocatechuate (PCA) 4,5-cleavage pathway during the degradation of lignin. The three-dimensional structure of the apo form of the wild-type enzyme was determined by X-ray crystallography, and the structure of the K66M mutant enzyme was determined in the presence of the substrate OMA. LigU is a homodimer requiring no cofactors or metal ions with a diaminopimelate epimerase structural fold, consisting of two domains with similar topologies. Each domain has a central α-helix surrounded by a β-barrel composed of antiparallel β-strands. The active site is at the cleft of the two domains. 1H nuclear magnetic resonance spectroscopy demonstrated that the enzyme catalyzes the exchange of the pro-S hydrogen at C5 of KCH with D2O during the isomerization reaction. Solvent–deuterium exchange experiments demonstrated that mutation of Lys-66 eliminated the isotope exchange at C5 and that mutation of C100 abolished exchange at C3. The positioning of these two residues in the active site of LigU is consistent with a reaction mechanism that is initiated by the abstraction of the pro-S hydrogen at C3 of OMA by the thiolate anion of Cys-100 and the donation of a proton at C5 of the proposed enolate anion intermediate by the side chain of Lys-66 to form the product KCH. The 1,3-proton transfer is suprafacial.

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Iron-sulfur (Fe-S) clusters have a key role in many biochemical processes and are essential for most life forms. Despite recent mechanistic advances in understanding the Fe-S cluster biosynthetic pathway, critical questions remain unresolved. Although human NFS1 and E. coli IscS share ∼60% sequence identity, NFS1 exhibits low activity and requires activation by the Friedreich’s ataxia protein frataxin (FXN) for in vivo function. Surprisingly, structures of the human complex reveal three distinct quaternary structures with one form exhibiting the same subunit interactions as IscS. An architectural switch model has been proposed in which evolutionarily lost interactions between NFS1 subunits results in the formation of low-activity architectures; FXN binding compensates for these lost interactions and facilitates a subunit rearrangement to activate the complex. Here, we used a structure and evolution-guided approach to identify three conserved residues proposed to weaken interactions between NFS1 subunits and transplanted these amino acids into IscS. Compared to native IscS, the engineered variant had a 4000-fold weaker dimer interface and diminished activity that correlated with the absence of the second catalytic subunit. Remarkably, the addition of the FXN homolog to the engineered variant stimulated the decay of the Cys-quinonoid pyridoxal 5’-phosphate intermediate, shifted IscS from the monomeric to dimeric form, and increased the cysteine desulfurase activity, reproducing results from the human system and supporting the architectural switch model. Overall, these studies indicate a weakening of the homodimeric interface was a key development during the evolution of the eukaryotic system and provide new insights into the role of FXN.