Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the β-aryl ether bond of lignin

Abstract

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate β-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGST_Nu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGST_Nu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr⁵¹, Tyr¹⁶⁶, and Tyr²²⁴) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the β-aryl ether bond) and Escherichia coli (which cannot break the β-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.

Keywords: Escherichia coli (E. coli); Novosphingobium aromaticivorans; Nu-class; bacterial metabolism; beta-aryl ether; deglutathionylation; enzyme mechanism; enzyme structure; glutathione S-transferases; lignin degradation.

Figure 1.

Breaking of the β-aryl ether (β-O-4) bond of GGE via the sphingomonad β-etherase pathway. Enzymes shown were identified in Sphingobium sp. SYK-6 (“Lig” enzymes (12–14)), Novosphingobium sp. MBES04 (GST3 (6)), or N. aromaticivorans DSM 12444 (NaGST_Nu; this work). The sphingomonads investigated in this work for GGE metabolism that are predicted to contain a given enzyme (6) are listed under the enzyme name. The α-, β-, and γ-carbons are labeled in the topmost GGE molecule. Erythro-GGE consists of the α(S)β(R) and α(R)β(S) stereoisomers; threo-GGE consists of the α(R)β(R) and α(S)β(S) stereoisomers. All chiral molecules are labeled with their chiralities.

Figure 2.

Cell densities and extracellular metabolite concentrations of representative N. aromaticivorans cultures grown in SMB containing 3 mm GGE (A–F) or 4 mm vanillate and 1.5 mm GGE (G–L). Data are shown for strains 12444Δ1879 (effective wildtype; A, B, G, and H), 12444Δ2595 (Saro_2595, which codes for NaGST_Nu, deleted from the genome of 12444Δ1879; C, D, I, and J), and 12444EcyghU (with E. coli yghU replacing Saro_2595 in the genome of 12444Δ1879; E, F, K, and L). The y-axes of H, J, and L use multiple scales. For comparison, cell densities for cultures grown in SMB containing only 4 mm vanillate are included in G, I, and K. Cell densities are in KU; for N. aromaticivorans, 1 KU ∼ 8 × 10⁶ cfu/ml (Table S4).

Figure 3.

Time courses for the reactions of N. aromaticivorans cell extracts with racemic β(R)- and β(S)-MPHPV. A, strain 12444Δ1879. B, strain 12444Δ2595. The red dotted line in B indicates the time at which recombinant NaGST_Nu and additional GSH were added to the reaction.

Figure 4.

Structure of NaGST_Nu (PDB 5UUO). A, domain structure of one subunit of the homodimer (with the open C-terminal configuration). The monomer contains a GST1 N-terminal (thioredoxin) domain (Val³⁹–Gly¹²⁹; green), a GST2 C-terminal domain (Ser¹³⁵–Leu²⁵⁷; maroon), and N terminus (Met¹–Pro³⁸; white) and C terminus (Val²⁵⁸–Phe²⁸⁸; gold) extensions. Atoms in Phe⁸², Tyr²²⁴, Lys²⁶², and Phe²⁸⁸ are shown as spheres. B, contacts between active-site residues and the GSH1 and GSH2 dithiol (60% occupancy; orange and cyan carbon atoms, respectively) and the GS-SG disulfide (40%; gray carbon atoms). NaGST_Nu residues are colored according to domain origin in A. Interactions involving protein residues are shown in black; those between GSH1 and GSH2 are in silver. Selected distances between interacting atoms are shown.

Figure 5.

Comparison of the region surrounding the active site in closely related Nu-class GSTs. A, alignment of subunits of NaGST_Nu (PDB code 5UUO) (blue, closed C-terminal configuration; white, open C-terminal configuration), EcYghU (PDB code 3C8E; orange), and SsYghU (PDB code 4MZW; green). Each subunit is labeled at its C terminus. Residues labeled in B–E with atoms shown as spheres are spatially conserved between the subunits and define a triangle used to approximate the area of each active-site channel opening. B, closed configuration of NaGST_Nu (channel opening ∼11 Ų). C, open configuration of NaGST_Nu (channel opening ∼18 Ų). D, EcYghU (channel opening ∼25 Ų). E, SsYghU (channel opening ∼25 Ų).

Figure 6.

Modeling of β(R)- and β(S)-GS-HPV into the NaGST_Nu and EcYghU active sites. The glutathione moiety of GS-HPV is modeled into the position occupied by GSH2 in the structures. Carbon atoms of GS-HPV are cyan. Those of GSH1 are orange. Coloring of NaGST_Nu residues is the same as in Fig. 4. EcYghU residues are colored the same as their NaGST_Nu analogues in Fig. 4. A and B show modeling into NaGST_Nu (PDB code 5UUO; closed C-terminal configuration). C and D show modeling into EcYghU (PDB code 3C8E). A and C show predicted interactions involving β(R)-GS-HPV. B and D show predicted interactions involving β(S)-GS-HPV. Residues Phe⁸² and Phe²⁸⁸ in NaGST_Nu and Arg²⁶⁰ and Asn²⁶² in EcYghU contribute to different internal dimensions of the active-site channels of NaGST_Nu and EcYghU, leading to different predicted orientations of the bound substrates between the enzymes. Fig. S7 shows space-filling models of the enzyme-substrate complexes.

Figure 7.

Proposed mechanism for NaGST_Nu-catalyzed cleavage of the thioether bond in β(R)-GS-HPV (top row) or β(S)-GS-HPV (bottom row). A and B, Thr⁵¹ and Asn⁵³ provide hydrogen bonds that stabilize a reactive GS1 thiolate anion, which attacks the GS- moiety of GS-HPV (occupying the active-site GSH2 position) to form a G1S-SG2 disulfide. C and D, rupture of the thioether bond is facilitated by formation of a transient enolate intermediate, which is stabilized by interactions between GS-HPV and Tyr¹⁶⁶ and Tyr²²⁴ of NaGST_Nu. E and F, capture of a solvent-derived proton by the carbanion collapses the enolate to form HPV. The hydroxyl group of the C-terminal Phe²⁸⁸ in the closed NaGST_Nu configuration provides a hydrogen bond that stabilizes the positioning of the substrate throughout the process.