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

ABSTRACT: 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 ?-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 ?-S-thioether linkages. Products of recombinant GSTs (LigE, LigP, and LigF) are ?-S-glutathionyl-?-keto-thioethers that are degraded by a ?-S-thioetherase (LigG). All three Lig GSTs produced the ketone product (?-S-glutathionyl-?-veratrylethanone) from an achiral side chain-truncated model substrate (?-guaiacyl-?-veratrylethanone). However, when ?-etherase assays were conducted with a racemic model substrate, ?-guaiacyl-?-veratrylglycerone, LigE- or LigP-catalyzed reactions yielded only one of two potential product (?-S-glutathionyl-?-veratrylglycerone) epimers, whereas the other diastereomer (differing in configuration at the ?-position (i.e. its ?-epimer)) was produced only in the LigF-catalyzed reaction. Thus, ?-etherase catalysis causes stereochemical inversion of the chiral center, converting a ?(R)-substrate to a ?(S)-product (LigE and LigP), and a ?(S)-substrate to a ?(R)-product (LigF). Further, LigG catalyzed glutathione-dependent ?-S-thioether cleavage with ?-S-glutathionyl-?-veratrylethanone and with ?(R)-configured ?-S-glutathionyl-?-veratrylglycerone but exhibited no or significantly reduced ?-S-thioether-cleaving activity with the ?(S)-epimer, demonstrating that LigG is a stereospecific ?-thioetherase. We therefore propose that multiple Lig enzymes are needed in this ?-aryl etherase pathway in order to cleave the racemic ?-ether linkages that are present in the backbone of the lignin polymer.

SUBMITTER: Gall DL

PROVIDER: S-EPMC3961688 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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

Gall Daniel L DL Kim Hoon H Lu Fachuang F Donohue Timothy J TJ Noguera Daniel R DR Ralph John J

The Journal of biological chemistry 20140207 12

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 β-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 stereosele ...[more]

PMID: 24509858

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Project description:The microbial conversion of lignin-derived aromatics is a promising strategy for the industrial utilization of this large biomass resource. However, efficient application requires an elucidation of the relevant transport and catabolic pathways. In Sphingobium sp. strain SYK-6, most of the enzyme genes involved in 5,5'-dehydrodivanillate (DDVA) catabolism have been characterized, but the transporter has not yet been identified. Here, we identified SLG_07710 (ddvK) and SLG_07780 (ddvR), genes encoding a putative major facilitator superfamily (MFS) transporter and MarR-type transcriptional regulator, respectively. A ddvK mutant of SYK-6 completely lost the capacity to grow on and convert DDVA. DdvR repressed the expression of the DDVA O-demethylase oxygenase component gene (ligXa), while DDVA acted as the gene inducer. A DDVA uptake assay was developed by employing this DdvR-controlled ligXa transcriptional regulatory system. A Sphingobium japonicum UT26S transformant expressing ddvK acquired DDVA uptake capacity, indicating that ddvK encodes the DDVA transporter. DdvK, probably requiring the proton motive force, was suggested to be a novel MFS transporter on the basis of the amino acid sequence similarity. Subsequently, we evaluated the effects of ddvK overexpression on the production of the DDVA metabolite 2-pyrone-4,6-dicarboxylate (PDC), a building block of functional polymers. A SYK-6 mutant of the PDC hydrolase gene (ligI) cultured in DDVA accumulated PDC via 5-carboxyvanillate and grew by utilizing 4-carboxy-2-hydroxypenta-2,4-dienoate. The introduction of a ddvK-expression plasmid into a ligI mutant increased the growth rate in DDVA and the amounts of DDVA converted and PDC produced after 48 h by 1.35- and 1.34-fold, respectively. These results indicate that enhanced transporter gene expression can improve metabolite production from lignin derivatives.IMPORTANCE The bioengineering of bacteria to selectively transport and metabolize natural substrates into specific metabolites is a valuable strategy for industrial-scale chemical production. The uptake of many substrates into cells requires specific transport systems, and so the identification and characterization of transporter genes are essential for industrial applications. A number of bacterial major facilitator superfamily transporters of aromatic acids have been identified and characterized, but many transporters of lignin-derived aromatic acids remain unidentified. The efficient conversion of lignin, an abundant but unutilized aromatic biomass resource, to value-added metabolites using microbial catabolism requires the characterization of transporters for lignin-derived aromatics. In this study, we identified the transporter gene responsible for the uptake of 5,5'-dehydrodivanillate, a lignin-derived biphenyl compound, in Sphingobium sp. strain SYK-6. In addition to characterizing its function, we applied this transporter gene to the production of a value-added metabolite from 5,5'-dehydrodivanillate.

Dataset Information

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

Publications

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

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