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

Abstract

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 β-aryl ether bond connecting most phenylpropanoid units of the lignin heteropolymer. Here, we tested three sphingomonads predicted to be capable of breaking the β-aryl ether bond of the dimeric aromatic compound guaiacylglycerol-β-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 β-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 β-aryl ether bond) and Escherichia coli (which cannot break the β-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.

Funneling aromatic products of chemically depolymerized lignin into 2-pyrone-4-6-dicarboxylic acid withNovosphingobium aromaticivorans

“…15 The present study addresses each of these issues using mutant strains of N. aromaticivorans DSM12444, a microbe naturally capable of degrading S, G, and H type aromatic compounds, as a well as lignin derived aromatic dimers. 25,33 We chose N. aromaticivorans DSM12444 due to its known or predicted ability to grow in the presence of multiple aromatic compounds, its suitability for genetic analysis and modifi-cation, its ability to co-metabolize aromatics in the presence of other organic compounds (such as sugars, which are another plentiful product of plant biomass degradation), and the potential to produce single valuable products using defined mutants.…”

“…4 Sphingomonad bacteria, such as N. aromaticivorans DSM12444, are known or predicted to be capable of breaking most of the linkages found between aromatic subunits in natural lignin in defined ways that yield predictable mono-aromatic products that can be further metabolized. 1,36 N. aromaticivorans, specifically, is known to be capable of degrading model aromatic dimers containing β-aryl-ether bonds 25 and its genome contains homologs of genes that code for the degradation of other aromatic dimers in Sphingobium sp. SYK-6.…”

“…A variant of N. aromaticivorans DSM12444 (strain 12444Δ1879) that lacks the gene Saro_1879 ( putative sacB; SARO_RS09410 in the recently reannotated genome in NCBI) 25 was used as a parent strain to create the deletion mutant strains 12444ΔligI (lacks gene Saro_2819; SARO_RS14300), 12444ΔdesCD (lacks the genes Saro_2864 and Saro_2865; SARO_RS14525 and SARO_RS14530), and 12444ΔligIΔdesCD (lacks genes Saro_2819, Saro_2864, and Saro_2865). All genetic modifications used a variant of the plasmid pk18mobsacB, 37 which contains sacB and a kanamycin resistance gene.…”

“…26 A complete genome sequence is available for this α-proteobacterium (GenBank NC_007794.1), and the organism is amenable to genetic and genomic techniques needed to test the role of individual genes in aromatic metabolism, and model, engineer, or improve its pathways. 25 Specifically, we show that by using a defined set of mutations, N. aromaticivorans can be engineered to simultaneously produce PDC from all three major types of plant-derived phenolic compounds (S, G, and H). In addition, we find that this organism can metabolize aromatics simultaneously with the use of other organic carbon sources (such as glucose), a feature that allows mutant strains to excrete compounds derived from the incomplete metabolism of the aromatics.…”

“…16 Some of these approaches require extensive metabolic re-routing and introduction of foreign pathways, 19,22 while others rely on a small number of mutations that redirect aromatic metabolism to the product of interest. 17,18 Here we report on the impact of gene deletions in the central aromatic catabolic pathways of Novosphingobium aromaticivorans DSM12444, an organism known to degrade aromatic compounds 24 and to break down interlinkages in lignin, 25 that allow it to funnel a large diversity of plant-derived phenolics into PDC, a potential bioplastic and epoxy adhesives precursor. 26 A complete genome sequence is available for this α-proteobacterium (GenBank NC_007794.1), and the organism is amenable to genetic and genomic techniques needed to test the role of individual genes in aromatic metabolism, and model, engineer, or improve its pathways.…”