Database Mining for Novel Bacterial β-Etherases, Glutathione-Dependent Lignin-Degrading Enzymes

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. beta-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of beta-O-4 aryl ether bonds. However, only a very limited number of these enzymes have been described and characterized so far. Using peptide pattern recognition (PPR) as well as phylogenetic analyses, 96 putatively novel beta-etherases have been identified, some even originating from bacteria outside the order Sphingomonadales. A set of 13 diverse enzymes was selected for biochemical characterization, and beta-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known beta-etherases. Moreover, conserved sequence motifs specific for either LigE- or LigF-type enzymes were deduced from multiple-sequence alignments and the PPR-derived peptides. In combination with structural information available for the beta-etherases LigE and LigF, insight into the potential structural and/or functional role of conserved residues within these sequence motifs is provided. Phylogenetic analyses further suggest the presence of additional bacterial enzymes with potential beta-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric beta-etherases. IMPORTANCE The use of biomass as a renewable source and replacement for crude oil for the provision of chemicals and fuels is of major importance for current and future societies. Lignin, the most abundant aromatic polymer in nature, holds promise as a renewable starting material for the generation of required aromatic structures. However, a controlled and selective lignin depolymerization to yield desired aromatic structures is a very challenging task. In this regard, bacterial beta-etherases are especially interesting, as they are able to cleave the most abundant bond type in lignin with high selectivity. With this study, we significantly expanded the toolbox of available beta-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further beta-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.