Structure and Chemical Reaction Mechanism of LigU, an Enzyme That Catalyzes an Allylic Isomerization in the Bacterial Degradation of Lignin.

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 ß-barrel composed of antiparallel ß-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.

Structure of human Fe-S assembly subcomplex reveals unexpected cysteine desulfurase architecture and acyl-ACP-ISD11 interactions.

In eukaryotes, sulfur is mobilized for incorporation into multiple biosynthetic pathways by a cysteine desulfurase complex that consists of a catalytic subunit (NFS1), LYR protein (ISD11), and acyl carrier protein (ACP). This NFS1-ISD11-ACP (SDA) complex forms the core of the iron-sulfur (Fe-S) assembly complex and associates with assembly proteins ISCU2, frataxin (FXN), and ferredoxin to synthesize Fe-S clusters. Here we present crystallographic and electron microscopic structures of the SDA complex coupled to enzyme kinetic and cell-based studies to provide structure-function properties of a mitochondrial cysteine desulfurase. Unlike prokaryotic cysteine desulfurases, the SDA structure adopts an unexpected architecture in which a pair of ISD11 subunits form the dimeric core of the SDA complex, which clarifies the critical role of ISD11 in eukaryotic assemblies. The different quaternary structure results in an incompletely formed substrate channel and solvent-exposed pyridoxal 5'-phosphate cofactor and provides a rationale for the allosteric activator function of FXN in eukaryotic systems. The structure also reveals the 4'-phosphopantetheine-conjugated acyl-group of ACP occupies the hydrophobic core of ISD11, explaining the basis of ACP stabilization. The unexpected architecture for the SDA complex provides a framework for understanding interactions with acceptor proteins for sulfur-containing biosynthetic pathways, elucidating mechanistic details of eukaryotic Fe-S cluster biosynthesis, and clarifying how defects in Fe-S cluster assembly lead to diseases such as Friedreich's ataxia. Moreover, our results support a lock-and-key model in which LYR proteins associate with acyl-ACP as a mechanism for fatty acid biosynthesis to coordinate the expression, Fe-S cofactor maturation, and activity of the respiratory complexes.