Structural Basis of Stereospecificity in the Bacterial Enzymatic Cleavage of β-Aryl Ether Bonds in Lignin

Abstract

Lignin is a combinatorial polymer comprising monoaromatic units that are linked via covalent bonds. Although lignin is a potential source of valuable aromatic chemicals, its recalcitrance to chemical or biological digestion presents major obstacles to both the production of second-generation biofuels and the generation of valuable coproducts from lignin's monoaromatic units. Degradation of lignin has been relatively well characterized in fungi, but it is less well understood in bacteria. A catabolic pathway for the enzymatic breakdown of aromatic oligomers linked via β-aryl ether bonds typically found in lignin has been reported in the bacterium Sphingobium sp. SYK-6. Here, we present x-ray crystal structures and biochemical characterization of the glutathione-dependent β-etherases, LigE and LigF, from this pathway. The crystal structures show that both enzymes belong to the canonical two-domain fold and glutathione binding site architecture of the glutathione S-transferase family. Mutagenesis of the conserved active site serine in both LigE and LigF shows that, whereas the enzymatic activity is reduced, this amino acid side chain is not absolutely essential for catalysis. The results include descriptions of cofactor binding sites, substrate binding sites, and catalytic mechanisms. Because β-aryl ether bonds account for 50–70% of all interunit linkages in lignin, understanding the mechanism of enzymatic β-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

Structural and functional divergence of GDAP1 from the glutathione S‐transferase superfamily

“…Structural predictions place the α‐loop just above S34 in the GST canonical active site and we predict that it completes a binding pocket that utilizes S34, the disordered helix α2, and surfaces on both the G‐ and H‐sites. Such an arrangement has been seen for other glutathione‐dependent enzymes 56 . It is also noteworthy that the position of the α‐loop is on the same face of the protein as the HD1 domain, suggesting that the α‐loop could play a role in interacting with the mitochondrial membrane or that the autoregulatory role proposed for the HD1 is mediated by an interaction between the HD1 and the α‐loop.…”

“…This is consistent with our biochemical observation that α‐loop deletion abrogates ethacrynic acid binding (Figure 2A). This positioning is not without precedent, however, as the lignin proteins, LigE and LigF, are glutathione activated beta‐etherases that contain a lid similar to the predicted structure of the α‐loop 56 . This model also predicts that a portion of the HD1 through residue 307 is in the proximity of the α‐loop consistent with a regulatory role for this domain 35 (Figure 5).…”

Alternatives for Chemical and Biochemical Lignin Valorization: Hot Topics from a Bibliometric Analysis of the Research Published During the 2000–2016 Period

“…The first β-etherases were already reported during the 1980s and Sphingobium sp. has been the most investigated case [403,404]. However, because of their high potential impact for lignin valorization, the search of novel enzymes from alternative organisms and their characterization have been reported [405].…”

Roles of two glutathioneS-transferases in the final step of the β-aryl ether cleavage pathway inSphingobiumsp. strain SYK-6

“…LigF and LigE/LigP attack (S)-MPHPV and (R)-MPHPV, subsequently producing (R)-GS-HPV and (S)-GS-HPV, respectively (14)(15)(16)(17)(18). Recently, X-ray crystal structures of LigE and LigF were determined, suggesting that LigE was most similar to the GSTFuA fungal class, and LigF was classified in a new structural class closely related to GSTFuAs or fungal Ure2p-like GST (19). LigF and LigE cleave the -aryl ether linkage through an S N 2 nucleophilic attack of GSH on the -carbon of the substrates (18,19).…”

“…Recently, X-ray crystal structures of LigE and LigF were determined, suggesting that LigE was most similar to the GSTFuA fungal class, and LigF was classified in a new structural class closely related to GSTFuAs or fungal Ure2p-like GST (19). LigF and LigE cleave the -aryl ether linkage through an S N 2 nucleophilic attack of GSH on the -carbon of the substrates (18,19). Another GST, LigG, catalyzes the cleavage of the thioether linkage in (R)-GS-HPV by transferring the (R)-GS-HPV glutathione moiety to another GSH molecule, producing achiral HPV and glutathione disulfide (GSSG) (14,16).…”