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

Biochemistry. 2019 Aug 20;58(33):3494-3503. doi: 10.1021/acs.biochem.9b00549. Epub 2019 Aug 9.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Bacterial Proteins / metabolism*
  • Carbohydrate Epimerases / metabolism
  • Catalytic Domain
  • Crystallography, X-Ray
  • Hydroxybenzoates / metabolism
  • Isomerases / metabolism*
  • Kinetics
  • Lignin / metabolism*
  • Models, Molecular*
  • Protein Conformation
  • Sphingomonadaceae / enzymology*


  • Bacterial Proteins
  • Hydroxybenzoates
  • protocatechuic acid
  • Lignin
  • Isomerases
  • Carbohydrate Epimerases