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.

Discovery of novel enzyme genes involved in the conversion of an arylglycerol-ß-aryl ether metabolite and their use in generating a metabolic pathway for lignin valorization.

Microbial conversions known as "biological funneling" have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained by chemical lignin depolymerization. ß-hydroxypropiovanillone (HPV) and its analogs can be obtained by chemoselective catalytic oxidation of lignin using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of arylglycerol-ß-aryl ether with zinc. Sphingobium sp. strain SYK-6 can degrade HPV generated by the catabolism of arylglycerol-ß-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a promising platform chemical. Therefore, production of PDC from HPV can be achieved using the HPV catabolic pathway. However, the pathway and genes involved in the catabolism of vanilloyl acetic acid (VAA) generated during HPV catabolism have not been investigated. In the present study, we isolated SLG_24960 (vceA), which encodes an enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate (vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida. The resulting engineered strain completely converted 1  mM HPV into PDC after 24  h. Our results suggest that the enzyme genes that are not required for the catabolic pathway in microorganisms but can be used for the conversion of target substrates are buried in microbial genomes. These genes are, thus, useful for designing metabolic pathways to produce value-added metabolites.

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS).

BACKGROUND: Producing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes. RESULTS: Two ß-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon-carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the ß-aryl ether bond. No cleavage of the carbon-carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 µL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics. CONCLUSIONS: A new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic ß-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol ß-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol ß-O-4 type of molecules with laccase and MnP.

Characterization of Lignin Extracted from Willow by Deep Eutectic Solvent Treatments.

Purity, morphology, and structural characterization of synthesized deep eutectic solvent (DES)-lignins (D6h, D9h, D12h, D18h, D24h) extracted from willow (Salix matsudana cv. Zhuliu) after treatment with a 1:10 molar ratio of choline chloride and lactic acid at 120 °C for 6, 9, 12, 18, and 24 h were carried out. The purity of DES-lignin was ~95.4%. The proportion of hydrogen (H) in DES-lignin samples increased from 4.22% to 6.90% with lignin extraction time. The DES-lignin samples had low number/weight average molecular weights (1348.1/1806.7 to 920.2/1042.5 g/mol, from D6h to D24h) and low particle sizes (702⁻400 nm). Atomic force microscopy (AFM) analysis demonstrated that DES-lignin nanoparticles had smooth surfaces and diameters of 200⁻420 nm. Syringyl (S) units were dominant, and total phenolic hydroxyl content and total hydroxyl content reached their highest values of 2.05 and 3.42 mmol·g-1 in D12h and D6h, respectively. ß-Aryl ether (ß-O-4) linkages were eliminated during DES treatment.