Structure and Reaction Mechanism of the LigJ Hydratase: An Enzyme Critical for the Bacterial Degradation of Lignin in the Protocatechuate 4,5-Cleavage Pathway

Abstract

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).

Metal- and Serine-Dependent Meta-Cleavage Product Hydrolases Utilize Similar Nucleophile-Activation Strategies

“…LigY shares notable mechanistic features with LigW, whose mechanism is a paradigm for COG2159 decarboxylases, and LigJ, a hydratase. , Shared features include the mode of substrate binding and the role of the conserved acidic metal ion ligand. In all three mechanisms, the substrate binds the metal ion in a bidentate fashion via a carboxylate and either a hydroxyl or oxo group on an adjacent carbon atom.…”

“…As noted above, the protonated Glu-282 in LigY is positioned to form the oxyanion hole together with the amide of Val-286 and, in the final steps of the reaction, could protonate the C2 oxido of CHPD as it is released from the enzyme (Figure ). In LigJ, Glu-284 does not appear to ligate the metal ion but is proposed to act as a base to activate water for attack at the substrate’s C3 . Finally, LigY and LigJ also share a hydrogen-bonding network involving His-223, Arg-234, Tyr-260, a water molecule, and the ligand’s C1 carboxylate (LigY numbering).…”

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

“…2-Pyrone-4,6-dicarboxylate (PDC, 1 ) was synthesized as previously described . The LigI hydrolase (UniProt entry O87170), LigJ hydratase (UniProt entry G2IQQ5), and LigK aldolase (UniProt entry G2IQQ8) were isolated and purified as previously described. − …”

“…8 LigJ hydrates KCH to (S)-4-carboxy-4-hydroxy-2-oxoadipate (CHA, 4). 9 Finally, LigK cleaves CHA into pyruvate (PYR, 5) and oxaloacetate (OAA, 6), which are then shuttled into the TCA cycle (Scheme 1). 10,11 Previously, we demonstrated that LigU isomerase catalyzes the conversion of OMA to KCH via a 1,3-allylic isomerization of the double bond from C4/C5 to C3/C4.…”

“…LigU isomerizes OMA into (3 Z )-2-keto-4-carboxy-3-hexendioate (KCH, 3 ) . LigJ hydrates KCH to ( S )-4-carboxy-4-hydroxy-2-oxoadipate (CHA, 4 ) . Finally, LigK cleaves CHA into pyruvate (PYR, 5 ) and oxaloacetate (OAA, 6 ), which are then shuttled into the TCA cycle (Scheme ).…”