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

Kontur, Wayne S.; Olmsted, Charles N.; Yusko, Larissa M.; Niles, Alyssa V.; Walters, Kevin A.; Beebe, Emily T.; Meulen, Kirk A. Vander; Karlen, Steven D.; Gall, Daniel L.; Noguera, Daniel R.; Donohue, Timothy J.

doi:10.1074/jbc.ra118.006548

Cited by 37 publications

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“…Sequence identities among LigF homologs vary between 56% and 96%, whereas NaLigF-2 is only 36% to 42% identical to the other LigF enzymes. Whereas classic ␤-etherases are homodimeric enzymes, Kontur et al recently described a novel heterodimeric type of ␤-etherases displaying R-selectivity (22). Here, sequence identities among the monomers of described heterodimeric ␤-etherases vary between 52% and 74%.…”

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confidence: 99%

“…In LigE-type ␤-etherases, sequence identities vary between 56% and 85%. LigF-type enzymes seem to be separated in two distinct subgroups, LigF homologs (including LigF, LigF-NA, LigF-NS, GST4, and NaLigF-1) and NaLigF-2 (22). Sequence identities among LigF homologs vary between 56% and 96%, whereas NaLigF-2 is only 36% to 42% identical to the other LigF enzymes.…”

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confidence: 99%

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

Voß

Heck

Schallmey

et al. 2020

Appl Environ Microbiol

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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.

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

Voß

Heck

Schallmey

et al. 2020

Appl Environ Microbiol

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“…[16] Glutathione (GSH) is a tripeptide used by the Lig enzymes E, P, F, and BaeAB (etherases), and Lig enzyme G and GST Nu (lyase). [16,17] This cofactor contains a cysteine (thiol) residue that performs a protein-assisted S N 2 reaction on an α-keto lignin dimer, forming a thioether. The thioether is then reduced by another glutathione resulting in the formation of a disulfide bond and liberation of an enolate that is protonated to form the ketone.…”

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“…The ability of small, diffusible redox mediators to penetrate and chemically attack the lignin network is a potential advantage relative to the Lig enzymes which are mostly limited to small lignin fragments. [17,19] Furthermore, these diffusible thiol-based redox carriers can theoretically be recycled using 2-electron/2-proton reduction of the disulfide product in an electrochemical cell ( Figure S28), analogous to the electrochemical oxidative mediators employed by Stahl and co-workers. [20] Herein, we first explore alkaline thiol-mediated cleavage of model β-aryl ether polymers into monomeric products, and then extend our studies to lignin extracted from poplar, and to other lignin-type linkages.…”

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Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin

et al. 2020

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Lignin may serve as a renewable feedstock for the production of chemicals and fuels if mild, scalable processes for its depolymerization can be devised. The use of small organic thiols represents a bioinspired strategy to cleave the β‐O‐4 bond, the most common linkage in lignin. In the present study, synthetic β‐O‐4 linked polymers were treated with organic thiols, yielding up to 90 % cleaved monomer products. Lignin extracted from poplar was also treated with organic thiols resulting in molecular weight reductions as high as 65 % (Mn) in oxidized lignin. Thiol‐based cleavage of other lignin linkages was also explored in small‐molecule model systems to uncover additional potential pathways by which thiols might depolymerize lignin. The success of thiol‐mediated cleavage on model dimers, polymers, and biomass‐derived lignin illustrates the potential utility of small redox‐active molecules to penetrate complex polymer matrices for depolymerization and subsequent valorization of lignin into fuels and chemicals.

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“…A classe das transferases apresentou 1085 sequências. O metabolismo da lignina é classicamente atribuído ao envolvimento de enzimas oxidativas (lacases, manganês peroxidases e lignina peroxidases), entretanto estudos recentes têm mostrado que alguns microorganismos expressam transferases que podem clivar ligações da lignina(CHEN et al, 2011;KONTUR et al, 2019). apenas nas porções C-terminal.…”

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Transcriptoma e expressão recombinante de peptidases do fungo Phanerochaete chrysosporium em Pichia pastoris

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metanol. A atividade enzimática dos clones contendo a construção pPICZαA-DN10508 (aspartil peptidase) mostraram que os clones 1 (16,00 ± 3,27 U/mL) ,2 (14,44 ± 1,12 U/mL), 11 (14,27± 0,315 U/mL), 4 (14,27 ± 0,086 U/mL) e 5 (14,15 ± 2,15 U/mL) obtiveram a maior atividade enzimática. A atividade enzimática dos clones contendo pPICZαA-DN7040 (cisteíno peptidase) não apresentaram resultados promissores. Nas condições avaliadas foi possível induzir a expressão de diferentes classes enzimáticas. A biblioteca de transcritos gerou mais de 33 mil sequências e através dela, foi possível selecionar e expressar duas peptidases (aspartil e cisteíno peptidases) para a expressão heteróloga. Palavras chave: Peptidases recombinantes, Aspartil peptidase, Cisteíno peptidase iii ABSTRACT SIMÕES, F.A.O. Trancriptome and recombinant peptidase expression from the fungus Phanerochaete chrysosporium in Pichia pastoris. 2019. 87p. Dissertation (Master). Faculdade de Ciências Farmacêuticas de Ribeirão Preto -Universidade de São Paulo, Ribeirão Preto, 2019.Fungi can be found in a variety of habitats and have great genetic diversity. These microorganisms produce a variety of enzymes and some of these may work under specific temperature and pH conditions. Phanerochaete chrysosporium can completely degrade lignin. Its genome possess a large repertoire of genes related to lignin metabolism including cytochrome p450, oxidoreductose and hydrolases, which includes peptidase enzymes. Peptidase enzymes are involved in various biological pathways and have industrial applications (textile and food industry). Our research group have described two native peptidases (aspartyl and cysteine peptidase) with great potential for industrial application. Recombinant protein production could provide an efficient way for enzyme production and purification. Pichia patoris expression system is widely used for recombinant protein expression, and it is described as efficient for both production and secretion of recombinant enzymes, it also is able to execute post-translational modifications. The aim of this study was to perform transcriptome analyses of the fungus Phanerochaete chrysosporium to identify and express two heterologous peptidase (aspartic and cysteine peptidase) in P. pastoris for biochemical and functional studies. P. chrysosporium was submitted to different bioprocesses supplemented with feather meal (1%), casein (1%), avicel (1%), or olive oil (1%). Enzymatic activities of peptidases, laccases, amylases, lipases, CMCases, xylanases and pectinases were determined. All activities were detected, except lipase and pectinase activities. Extracts supplemented with feather meal were purified by ion exchange chromatography and the N-terminal sequence of aspartic and cysteine peptidases were determined by Edman degradation. The mycelium was used for RNA extraction and cDNA synthesis. A transcript library was built using cDNA sequences, which generated 33840 sequences. The library was analyzed by UNIPROT and 7327 sequences were identified (52% non-enzymes and 4...

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

Cited by 37 publications

References 53 publications

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

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

Nucleophilic Thiols Reductively Cleave Ether Linkages in Lignin Model Polymers and Lignin

Transcriptoma e expressão recombinante de peptidases do fungo Phanerochaete chrysosporium em Pichia pastoris

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