A heterodimeric glutathione S-transferase that stereospecifically breaks lignin's ß(R)-aryl ether bond reveals the diversity of bacterial ß-etherases.

Lignin is a heterogeneous polymer of aromatic subunits that is a major component of lignocellulosic plant biomass. Understanding how microorganisms deconstruct lignin is important for understanding the global carbon cycle and could aid in developing systems for processing plant biomass into valuable commodities. Sphingomonad bacteria use stereospecific glutathione S-transferases (GSTs) called ß-etherases to cleave the ß-aryl ether (ß-O-4) bond, the most common bond between aromatic subunits in lignin. Previously characterized bacterial ß-etherases are homodimers that fall into two distinct GST subclasses: LigE homologues, which cleave the ß(R) stereoisomer of the bond, and LigF homologues, which cleave the ß(S) stereoisomer. Here, we report on a heterodimeric ß-etherase (BaeAB) from the sphingomonad Novosphingobium aromaticivorans that stereospecifically cleaves the ß(R)-aryl ether bond of the di-aromatic compound ß-(2-methoxyphenoxy)-γ-hydroxypropiovanillone (MPHPV). BaeAB's subunits are phylogenetically distinct from each other and from other ß-etherases, although they are evolutionarily related to LigF, despite the fact that BaeAB and LigF cleave different ß-aryl ether bond stereoisomers. We identify amino acid residues in BaeAB's BaeA subunit important for substrate binding and catalysis, including an asparagine that is proposed to activate the GSH cofactor. We also show that BaeAB homologues from other sphingomonads can cleave ß(R)-MPHPV and that they may be as common in bacteria as LigE homologues. Our results suggest that the ability to cleave the ß-aryl ether bond arose independently at least twice in GSTs and that BaeAB homologues may be important for cleaving the ß(R)-aryl ether bonds of lignin-derived oligomers in nature.

Novosphingobium aromaticivorans uses a Nu-class glutathione S-transferase as a glutathione lyase in breaking the ß-aryl ether bond of lignin.

As a major component of plant cell walls, lignin is a potential renewable source of valuable chemicals. Several sphingomonad bacteria have been identified that can break the ß-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the ß-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-ß-guaiacyl ether (GGE) and found that Novosphingobium aromaticivorans metabolizes GGE at one of the fastest rates thus far reported. After the ether bond of racemic GGE is broken by replacement with a thioether bond involving glutathione, the glutathione moiety must be removed from the resulting two stereoisomers of the phenylpropanoid conjugate ß-glutathionyl-γ-hydroxypropiovanillone (GS-HPV). We found that the Nu-class glutathione S-transferase NaGSTNu is the only enzyme needed to remove glutathione from both (R)- and (S)-GS-HPV in N. aromaticivorans We solved the crystal structure of NaGSTNu and used molecular modeling to propose a mechanism for the glutathione lyase (deglutathionylation) reaction in which an enzyme-stabilized glutathione thiolate attacks the thioether bond of GS-HPV, and the reaction proceeds through an enzyme-stabilized enolate intermediate. Three residues implicated in the proposed mechanism (Thr51, Tyr166, and Tyr224) were found to be critical for the lyase reaction. We also found that Nu-class GSTs from Sphingobium sp. SYK-6 (which can also break the ß-aryl ether bond) and Escherichia coli (which cannot break the ß-aryl ether bond) can also cleave (R)- and (S)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione S-transferases.

In Vitro Enzymatic Depolymerization of Lignin with Release of Syringyl, Guaiacyl, and Tricin Units.

New environmentally sound technologies are needed to derive valuable compounds from renewable resources. Lignin, an abundant polymer in terrestrial plants comprised predominantly of guaiacyl and syringyl monoaromatic phenylpropanoid units, is a potential natural source of aromatic compounds. In addition, the plant secondary metabolite tricin is a recently discovered and moderately abundant flavonoid in grasses. The most prevalent interunit linkage between guaiacyl, syringyl, and tricin units is the ß-ether linkage. Previous studies have shown that bacterial ß-etherase pathway enzymes catalyze glutathione-dependent cleavage of ß-ether bonds in dimeric ß-ether lignin model compounds. To date, however, it remains unclear whether the known ß-etherase enzymes are active on lignin polymers. Here we report on enzymes that catalyze ß-ether cleavage from bona fide lignin, under conditions that recycle the cosubstrates NAD+ and glutathione. Guaiacyl, syringyl, and tricin derivatives were identified as reaction products when different model compounds or lignin fractions were used as substrates. These results demonstrate an in vitro enzymatic system that can recycle cosubstrates while releasing aromatic monomers from model compounds as well as natural and engineered lignin oligomers. These findings can improve the ability to produce valuable aromatic compounds from a renewable resource like lignin.IMPORTANCE Many bacteria are predicted to contain enzymes that could convert renewable carbon sources into substitutes for compounds that are derived from petroleum. The ß-etherase pathway present in sphingomonad bacteria could cleave the abundant ß-O-4-aryl ether bonds in plant lignin, releasing a biobased source of aromatic compounds for the chemical industry. However, the activity of these enzymes on the complex aromatic oligomers found in plant lignin is unknown. Here we demonstrate biodegradation of lignin polymers using a minimal set of ß-etherase pathway enzymes, the ability to recycle needed cofactors (glutathione and NAD+) in vitro, and the release of guaiacyl, syringyl, and tricin as depolymerized products from lignin. These observations provide critical evidence for the use and future optimization of these bacterial ß-etherase pathway enzymes for industrial-level biotechnological applications designed to derive high-value monomeric aromatic compounds from lignin.

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

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 ß-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 ß-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 ß-aryl ether bonds account for 50-70% of all interunit linkages in lignin, understanding the mechanism of enzymatic ß-aryl ether cleavage has significant potential for informing ongoing studies on the valorization of lignin.

Structural and Biochemical Characterization of the Early and Late Enzymes in the Lignin ß-Aryl Ether Cleavage Pathway from Sphingobium sp. SYK-6.

There has been great progress in the development of technology for the conversion of lignocellulosic biomass to sugars and subsequent fermentation to fuels. However, plant lignin remains an untapped source of materials for production of fuels or high value chemicals. Biological cleavage of lignin has been well characterized in fungi, in which enzymes that create free radical intermediates are used to degrade this material. In contrast, a catabolic pathway for the stereospecific cleavage of ß-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. ß-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic ß-aryl ether (ß-ether) cleavage is important for future efforts to biologically process lignin and its breakdown products. The crystal structures and biochemical characterization of the NAD-dependent dehydrogenases (LigD, LigO, and LigL) and the glutathione-dependent lyase LigG provide new insights into the early and late enzymes in the ß-ether degradation pathway. We present detailed information on the cofactor and substrate binding sites and on the catalytic mechanisms of these enzymes, comparing them with other known members of their respective families. Information on the Lig enzymes provides new insight into their catalysis mechanisms and can inform future strategies for using aromatic oligomers derived from plant lignin as a source of valuable aromatic compounds for biofuels and other bioproducts.

Stereochemical features of glutathione-dependent enzymes in the Sphingobium sp. strain SYK-6 ß-aryl etherase pathway.

Glutathione-dependent enzymes play important protective, repair, or metabolic roles in cells. In particular, enzymes in the glutathione S-transferase (GST) superfamily function in stress responses, defense systems, or xenobiotic detoxification. Here, we identify novel features of bacterial GSTs that cleave ß-aryl ether bonds typically found in plant lignin. Our data reveal several original features of the reaction cycle of these GSTs, including stereospecific substrate recognition and stereoselective formation of ß-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are ß-S-glutathionyl-α-keto-thioethers that are degraded by a ß-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (ß-S-glutathionyl-α-veratrylethanone) from an achiral side chain-truncated model substrate (ß-guaiacyl-α-veratrylethanone). However, when ß-etherase assays were conducted with a racemic model substrate, ß-guaiacyl-α-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (ß-S-glutathionyl-α-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the ß-position (i.e. its ß-epimer)) was produced only in the LigF-catalyzed reaction. Thus, ß-etherase catalysis causes stereochemical inversion of the chiral center, converting a ß(R)-substrate to a ß(S)-product (LigE and LigP), and a ß(S)-substrate to a ß(R)-product (LigF). Further, LigG catalyzed glutathione-dependent ß-S-thioether cleavage with ß-S-glutathionyl-α-veratrylethanone and with ß(R)-configured ß-S-glutathionyl-α-veratrylglycerone but exhibited no or significantly reduced ß-S-thioether-cleaving activity with the ß(S)-epimer, demonstrating that LigG is a stereospecific ß-thioetherase. We therefore propose that multiple Lig enzymes are needed in this ß-aryl etherase pathway in order to cleave the racemic ß-ether linkages that are present in the backbone of the lignin polymer.

A group of sequence-related sphingomonad enzymes catalyzes cleavage of ß-aryl ether linkages in lignin ß-guaiacyl and ß-syringyl ether dimers.

Lignin biosynthesis occurs via radical coupling of guaiacyl and syringyl hydroxycinnamyl alcohol monomers (i.e., "monolignols") through chemical condensation with the growing lignin polymer. With each chain-extension step, monolignols invariably couple at their ß-positions, generating chiral centers. Here, we report on activities of bacterial glutathione-S-transferase (GST) enzymes that cleave ß-aryl ether bonds in lignin dimers that are composed of different monomeric units. Our data reveal that these sequence-related enzymes from Novosphingobium sp. strain PP1Y, Novosphingobium aromaticivorans strain DSM12444, and Sphingobium sp. strain SYK-6 have conserved functions as ß-etherases, catalyzing cleavage of each of the four dimeric α-keto-ß-aryl ether-linked substrates (i.e., guaiacyl-ß-guaiacyl, guaiacyl-ß-syringyl, syringyl-ß-guaiacyl, and syringyl-ß-syringyl). Although each ß-etherase cleaves ß-guaiacyl and ß-syringyl substrates, we have found that each is stereospecific for a given ß-enantiomer in a racemic substrate; LigE and LigP ß-etherase homologues exhibited stereospecificity toward ß(R)-enantiomers whereas LigF and its homologues exhibited ß(S)-stereospecificity. Given the diversity of lignin's monomeric units and the racemic nature of lignin polymers, we propose that bacterial catabolic pathways have overcome the existence of diverse lignin-derived substrates in nature by evolving multiple enzymes with broad substrate specificities. Thus, each bacterial ß-etherase is able to cleave ß-guaiacyl and ß-syringyl ether-linked compounds while retaining either ß(R)- or ß(S)-stereospecificity.

Biochemical transformation of lignin for deriving valued commodities from lignocellulose.

The biochemical properties of lignin present major obstacles to deriving societally beneficial entities from lignocellulosic biomass, an abundant and renewable feedstock. Similar to other biopolymers such as polysaccharides, polypeptides, and ribonucleic acids, lignin polymers are derived from multiple types of monomeric units. However, lignin's renowned recalcitrance is largely attributable to its racemic nature and the variety of covalent inter-unit linkages through which its aromatic monomers are linked. Indeed, unlike other biopolymers whose monomers are consistently inter-linked by a single type of covalent bond, the monomeric units in lignin are linked via non-enzymatic, combinatorial radical coupling reactions that give rise to a variety of inter-unit covalent bonds in mildly branched racemic polymers. Yet, despite the chemical complexity and stability of lignin, significant strides have been made in recent years to identify routes through which valued commodities can be derived from it. This paper discusses emerging biological and biochemical means through which degradation of lignin to aromatic monomers can lead to the derivation of commercially valuable products.