Beta-Etherase and benzoyl-CoA pathway enzymes mediate biodegradation of lignin-related aromatic compounds

Lignocellulose is composed of Earth's three most abundant biopolymers: cellulose, hemi-celluloses, and lignin. Lignin's recalcitrance presents major obstacles to deriving commercially desirable entities from lignocellulosic biomass and is largely attributable to the various types of racemic units with several types of inter-unit covalent bonds through which the polymer's aromatic monomers (coniferyl alcohol that produces guaiacyl units and sinapyl alcohol that produces syringyl units) are linked by combinatorial radical coupling reactions. Cleavage of the beta-ether bonds, the most prominent type of inter-unit linkage, is thus crucial to lignin degradation processes. The use of purified recombinant "Lig" enzymes from Sphingobium sp. strain SYK-6, Novo-sphingobium sp. strain PP1Y, and Novosphingobium aromaticivorans strain DSM12444 in in vitro biochemical assays with glutathione and lignin model beta-ether-linked aromatic dimers as co-substrates revealed that each organism possesses both the beta(R)- and beta( S)-specific beta-etherase enzymes that are required to cleave the racemic beta-ether linkages found in lignin. I also found that each Lig enzyme exhibits beta-etherase activity with all four types of beta-ether-linked dimer substructures found in nature: guaiacyl-beta-guaiacyl, syringyl-beta-guaiacyl, guaiacyl-beta-syringyl, and syringyl-beta-syringyl. Further investigation of the Lig enzymes from strain SYK-6 indicates that beta-etherase catalysis involves cleavage of the beta-ether bond resulting in a glutathione-conjugated beta-S-thioether linkage and that this reaction causes inversion of the beta-chirality ( i.e., conversion of beta(R)-substrates to beta(S)-products and beta(S)-substrates to beta(R)-products). In sum, these findings demonstrate that beta-etherases are strictly stereoselective with respect to the beta-configuration of their products, either beta( R)- or beta(S)-stereospecific amongst racemic substrates, and yet, non-specific with regard to the substrate's guaiacyl/syringyl composition. In a parallel study, I also found that metabolism of meta-hydroxy-aromatic acids (protocatechuate and m-hydroxy-benzoate) in the bacterium Rhodopseudomonas palustris can be induced via the benzoyl-CoA pathway by metabolism of other pathway substrates ( e.g., benzoate). Although meta-hydroxy-aromatic acids were poor sole carbon sources, they were readily degraded in the presence of benzoate. R. palustris hbaBCD and badDEFG mutant phenotypes demonstrated that dehydroxylase HbaBCD and dearomatizing enzyme BadDEFG are required for protocatechuate m-hydroxy-benzoate metabolism, respectively. These results indicate that meta-substituted monoaromatic compounds, similar to those likely to be derived from lignin via beta-ether cleavage, can be reductively transformed in vivo via benzoyl-CoA pathway enzymes, yielding potentially valuable commodities as pathway intermediates.