Halohydrin dehalogenases (HHDHs) are of biotechnological interest due to their promiscuous epoxide ring-opening activity with a set of negatively charged nucleophiles, enabling the formation of C−C, C−N, or C−O bonds. The recent discovery of HHDH-specific sequence motifs aided the identification of a large number of halohydrin dehalogenases from public sequence databases, enlarging the biocatalytic toolbox substantially. During the characterization of 17 representatives of these phylogenetically diverse enzymes, one HHDH, namely HheG from Ilumatobacter coccineus, was identified to convert cyclic epoxide substrates. The enzyme exhibits significant activity in the azidolysis of cyclohexene oxide and limonene oxide with turnover numbers of 7.8 and 44 s −1 , respectively. As observed for other HHDHs, the cyanide-mediated epoxide ring-opening proceeded with lower rates. Wild-type HheG displays modest enantioselectivity, as the resulting azido-and cyanoalcohols of cyclohexene oxide ring-opening were obtained in 40% enantiomeric excess. These biocatalytic findings were further complemented by the crystal structure of the enzyme refined to 2.3 Å. Analysis of HheG's structure revealed a large open cleft harboring the active site. This is in sharp contrast to other known HHDH structures and aids in explaining the special substrate scope of HheG.

Regioselective methods to establish carbon–halide bonds are still rare, although halogenation is considered as a commonly used methodology for the functionalization of organic compounds. The incorporation of halogen substituents by organic synthesis usually requires hazardous conditions, shows poor regioselectivity and results in the formation of unwanted byproducts. In addition, halogenation by electrophilic aromatic substitution (SEAr) obeys distinct rules depending on electron‐withdrawing or ‐donating groups already present in the aromatic ring. We employed the tryptophan‐7‐halogenase RebH for regioselective enzymatic halogenation to overcome these limitations. In combination with a tryptophan synthase, an array of C5‐ and C6‐substituted tryptophan derivatives was synthesized and halogenated by RebH. The halogenase is able override these directing effects and halogenates at the electronically unfavored C7‐meta‐position, even in presence of ortho/para‐directing groups.

The combination of the biocatalytic halogenation of l‐tryptophan with subsequent chemocatalytic Suzuki–Miyaura cross‐coupling reactions leads to the modular synthesis of an array of C5, C6, or C7 aryl‐substituted tryptophan derivatives. In a three‐step one‐pot reaction, the bromo substituent is initially incorporated regioselectively by immobilized tryptophan 5‐, 6‐, or 7‐halogenases, respectively, with concomitant cofactor regeneration. The halogenation proceeds in aqueous media at room temperature in the presence of NaBr and O2. After the separation of the biocatalyst by filtration, a Pd catalyst, base, and boronic acid are added to the aryl halide formed in situ to effect direct Suzuki–Miyaura cross‐coupling reactions followed by tert‐butoxycarbonyl (Boc) protection. After a single purification step, different Boc‐protected aryl tryptophan derivatives are obtained that can, for example, be used for peptide or peptidomimetic synthesis.

Lignin is the major aromatic biopolymer in nature, and it is considered a valuable feedstock for the future supply of aromatics. Hence, its valorisation in biorefineries is of high importance, and various chemical and enzymatic approaches for lignin depolymerisation have been reported. Among the enzymes known to act on lignin, β-etherases offer the possibility for a selective cleavage of the β-O-4 aryl ether bonds present in lignin. These enzymes, together with glutathione lyases, catalyse a reductive, glutathione-dependent ether bond cleavage displaying high stereospecificity. β-Etherases and glutathione lyases both belong to the superfamily of glutathione transferases, and several structures have been solved recently. Additionally, different approaches for their application in lignin valorisation have been reported in the last years. This review gives an overview on the current knowledge on β-etherases and glutathione lyases, their biochemical and structural features, and critically discusses their potential for application in biorefineries.

Lignin is the most abundant aromatic polymer in nature and a promising renewable source for the provision of aromatic platform chemicals and biofuels. β-Etherases are enzymes with a promising potential for application in lignin depolymerization due to their selectivity in the cleavage of β-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 β-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 β-etherase activity was confirmed for all of them. Some enzymes displayed up to 3-fold higher activity than previously known β-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 β-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 β-etherase activity outside the classical LigE- and LigF-type enzymes as well as the recently described heterodimeric β-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 β-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 β-etherases for application in lignin depolymerization and discovered more active as well as diverse enzymes than previously known. Moreover, the identification of further β-etherases by sequence database mining in the future will be facilitated considerably through our deduced etherase-specific sequence motifs.