6dxs
From Proteopedia
Crystal structure of the LigJ hydratase E284Q mutant substrate complex with (3Z)-2-keto-4-carboxy-3-hexenedioate
Structural highlights
FunctionLIGJ_SPHSK Contributes to the degradation of lignin at the level of the protocatechuate 4,5-cleavage pathway. Catalyzes the hydration of the double bond of (3Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to (4S)-4-carboxy-4-hydroxy-2-oxoadipate (CHA, also named (2S)-2-hydroxy-4-oxobutane-1,2,4-tricarboxylate) (PubMed:29658701, PubMed:30207699). Is involved in the catabolism of both vanillate and syringate (PubMed:11092855).[1] [2] [3] Publication Abstract from PubMedLigJ from the soil bacterium Sphingobium sp. SYK-6 catalyzes the reversible hydration of (3Z)-2-keto-4-carboxy-3-hexenedioate (KCH) to 4-carboxy-4-hydroxy-2-oxoadipate (CHA) in the degradation of lignin in the protocatechuate (PCA) 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 solved in the presence (PDB id: 6DXQ) and absence of the product CHA (PDB id: 6DWV). The protein folds as a distorted (beta/alpha)8-barrel and a single zinc ion is bound in the active site at the C-terminal end of the central beta-barrel. The product CHA is ligated to the zinc ion in the active site via the displacement of the side-chain carboxylate of Glu-284 and 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 mutant E284Q 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 id: 6DXS). Based on 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 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 and 2.6 x 106 M-1 s-1 for kcat and kcat/Km, respectively. 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 TN, Mabanglo MF, Raushel FM Biochemistry. 2018 Sep 12. doi: 10.1021/acs.biochem.8b00713. PMID:30207699[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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