Sphingobium sp. SYK-6 is a Gram-negative soil bacterium that contributes to the degradation of lignin. Lignin provides structural support and protection to plants as a complex aromatic heteropolymer. The lignin degradation pathway of guaiacyl moieties leads to the intermediate, protocatechuate (PCA), which is further degraded via the 4,5-cleavage pathway in which PCA is ultimately metabolized to pyruvate and oxaloacetate. In this pathway, LigI has been shown to catalyze the hydrolysis of 2-pyrone-4,6-dicarboxylate to (4 E)-oxalomesaconate (OMA). Here we have demonstrated, using H andC nuclear magnetic resonance spectroscopy, that LigU catalyzes the isomerization of the double bond between C4 and C5 in (4 E)-OMA to (3 Z)-2-keto-4-carboxy-3-hexenedioate (KCH), where the double bond has migrated to be between C3 and C4 via a 1,3-allylic isomerization. LigU is most closely related in amino acid sequence to methylaconitate isomerase (PrpF) from Shewanella oneidensis and methylitaconate-Δ-isomerase (Mii) from Eubacterium barkeri. The kinetic constants for the isomerization of OMA to KCH by LigU at pH 8.0 were determined to be 1300 ± 120 s and (7.7 ± 1.5) × 10 M s for k and k/ K, respectively. We have also shown that the product of the LigU-catalyzed reaction is the preferred substrate for the LigJ hydratase. In this reaction, LigJ catalyzes the hydration of KCH to 4-carboxy-4-hydroxy-2-oxoadipate.
LigJ from the soil bacterium Sphingobium sp. SYK-6 catalyzes the reversible hydration of (3 Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) in the degradation of lignin in the protocatechuate 4,5-cleavage pathway. LigJ is a member of the amidohydrolase superfamily and an enzyme in cog2159. The three-dimensional crystal structure of wild-type LigJ was determined in the presence [Protein Data Bank (PDB) entry 6DXQ ] and absence of the product CHA (PDB entry 6DWV ). The protein folds as a distorted (β/α)-barrel, and a single zinc ion is bound in the active site at the C-terminal end of the central β-barrel. The product CHA is ligated to the zinc ion in the active site via the displacement of a single water molecule from the coordination shell of the metal center in LigJ. The product-bound structure reveals that the enzyme catalyzes the hydration of KCH with the formation of a chiral center at C4 with S stereochemistry. The E284Q mutant was unable to catalyze the hydration of KCH to CHA, and the structure of this mutant was determined in the presence of the substrate KCH (PDB entry 6DXS ). On the basis of the structure of LigJ in the presence of KCH and CHA, it is proposed that the side chain carboxylate of Glu-284 functions as a general base in the abstraction of a proton from a bound water molecule for nucleophilic attack at C4 of the substrate. The reaction is facilitated by the delocalization of the negative charge to the metal center via the carbonyl group at C2 of the substrate. C3 of the substrate is subsequently protonated by Glu-284 functioning as a general acid. The overall reaction occurs by the syn addition of water to the double bond between C4 and C3 of the substrate KCH. The kinetic constants for the hydration of KCH to CHA by LigJ at pH 8.0 are 25 s ( k) and 2.6 × 10 M s ( k/ K).
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.