Publications

• Structure and reaction mechanism of the LigJ hydratase: an enzyme critical for the bacterial degradation of lignin in the protocatechuate 4,5-cleavage pathway.

  Hogancamp T.N., Mabanglo M.F., Raushel F.M.

  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 superfamil ... >> More

  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)₈-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^-1 ( k_cat) and 2.6 × 10⁶ M^-1 s^-1 ( k_cat/ K_m). << Less

  Biochemistry 57:5841-5850(2018) [PubMed] [EuropePMC]

• Functional annotation of LigU as a 1,3-allylic isomerase during the degradation of lignin in the protocatechuate 4,5-cleavage pathway from the soil bacterium Sphingobium sp. SYK-6.

  Hogancamp T.N., Raushel F.M.

  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 (PC ... >> More

  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 ¹H and ¹³C 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^-1 and (7.7 ± 1.5) × 10⁶ M^-1 s^-1 for k_cat and k_cat/ K_m, 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. << Less

  Biochemistry 57:2837-2845(2018) [PubMed] [EuropePMC]

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