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

Abstract

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.

Regulation of vanillate and syringate catabolism by a MarR-type transcriptional regulator DesR in Sphingobium sp. SYK-6

“…SYK-6. The catabolic pathways proposed in our previous study 12,27 have been updated based on a recent report 49 . Enzymes: LigM, vanillate/3MGA O -demethylase; LigA and LigB, small and large subunits, respectively, of PCA 4,5-dioxygenase; LigC, CHMS dehydrogenase; LigI, PDC hydrolase; LigU, OMA delta-isomerase; LigJ, KCH hydratase; LigK, CHA aldolase; DesA, syringate O -demethylase; DesZ, 3MGA 3,4-dioxygenase; DesB, gallate dioxygenase.…”

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

“…The gene was expressed in Escherichia coli BL21(DE3) (EMD Millipore), and the protein purified as previously described. 8 The mutants S21A, K66Q, K66M, C100S, S254A, K257M, and H281N were constructed using the Agilent QuikChange protocol, and the primers are listed in Table S1. The mutant enzymes were purified as described previously for the wild-type enzyme.…”

“…The mutant enzymes were purified as described previously for the wild-type enzyme. 8 To improve the purity of the enzymes used in the crystallization trials, sizeexclusion chromatography was added to the purification protocol of LigU. 8 Protein was injected onto a HiLoad 16/ 600 Superdex 200 size-exclusion chromatography column (GE Healthcare) equilibrated in 20 mM HEPES/KOH (pH 7.9) and eluted using a flow rate of 0.8 mL/min.…”

“…8 To improve the purity of the enzymes used in the crystallization trials, sizeexclusion chromatography was added to the purification protocol of LigU. 8 Protein was injected onto a HiLoad 16/ 600 Superdex 200 size-exclusion chromatography column (GE Healthcare) equilibrated in 20 mM HEPES/KOH (pH 7.9) and eluted using a flow rate of 0.8 mL/min. The fractions were checked for purity by Mini-PROTEAN TGX stain-free sodium dodecyl sulfate−polyacrylamide gel electrophoresis (Bio-Rad).…”

“…7 LigU isomerizes OMA into (3Z)-2-keto-4-carboxy-3-hexendioate (KCH, 3). 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).…”