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.
Highlights
In contrast to known fungal systems, the bacterium Sphingobium sp. strain SYK-6 possesses an enzymatic route to the breakdown of lignin-derived components that is stereospecific and independent of chemical mediators and requires common cellular cofactors, such as pyridine nucleotides and glutathione
A conserved catalytic serine is a characteristic of the Theta class, Zeta class, and some bacterial GSTs [15], but there is evidence of GSTs from the bacteria P. mirabilis, Ochrubactrum anthropi, and E. coli in which this active site serine is not critical for catalytic activity [67,68,69]
Based on the data presented here and support from previous studies, it is clear that the active site serine is not responsible for the direct activation of the thiolate anion by deprotonation or perturbation of the pKa of the bound glutathione, it may be active in binding GSH in the active site, orienting the sulfhydryl group of GSH in the catalytic step, or stabilization of the transition state
Summary
Achiral derivative ␥-hydroxypropiovanillone, which serves as the growth substrate for strain SYK-6 [6, 10] (Fig. 1). It has been reported that these GST family member enzymes have the ability to work with lignin-derived materials in vitro [11, 12]. GSTs with Ͼ40% sequence identity are traditionally considered to be in the same class, whereas proteins of different classes have typically Ͻ25% protein sequence identity [15] These classifications are based on a number of other considerations, including structure, function, and biochemical properties [15]. We describe three protein crystal structures and provide the corresponding biochemical data for the LigE and LigF enzymes involved in the -ether cleavage step of the Sphingobium sp. The modest structural homology of these two enzymes highlights the fitness adaptation afforded in this and probably other microbial catabolic pathways that can degrade lignin-derived materials, required for enzymatic degradation of such racemic products. Because lignin is the most abundant aromatic polymer in nature, this study informs broader lignin valorization efforts that will enable the development of efficient pathways for the conversion of lignin into renewable aromatics with applications in advanced biofuels and chemicals [23]
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