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

ABSTRACT: 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 ?-aryl ether units that are found in lignin has been identified in Sphingobium sp. SYK-6 bacteria. ?-Aryl ether units are typically abundant in lignin, corresponding to 50-70% of all of the intermonomer linkages. Consequently, a comprehensive understanding of enzymatic ?-aryl ether (?-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 ?-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.

SUBMITTER: Pereira JH

PROVIDER: S-EPMC4858972 | biostudies-literature | 2016 May

REPOSITORIES: biostudies-literature

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Roles of two glutathione S-transferases in the final step of the β-aryl ether cleavage pathway in Sphingobium sp. strain SYK-6.

Project description:Sphingobium sp. strain SYK-6 is an alphaproteobacterial degrader of lignin-derived aromatic compounds, which can degrade all the stereoisomers of β-aryl ether-type compounds. SYK-6 cells convert four stereoisomers of guaiacylglycerol-β-guaiacyl ether (GGE) into two enantiomers of α-(2-methoxyphenoxy)-β-hydroxypropiovanillone (MPHPV) through GGE α-carbon atom oxidation by stereoselective Cα-dehydrogenases encoded by ligD, ligL, and ligN. The ether linkages of the resulting MPHPV enantiomers are cleaved by stereoselective glutathione (GSH) S-transferases (GSTs) encoded by ligF, ligE, and ligP, generating (βR/βS)-α-glutathionyl-β-hydroxypropiovanillone (GS-HPV) and guaiacol. To date, it has been shown that the gene products of ligG and SLG_04120 (ligQ), both encoding GST, catalyze GSH removal from (βR/βS)-GS-HPV, forming achiral β-hydroxypropiovanillone. In this study, we verified the enzyme properties of LigG and LigQ and elucidated their roles in β-aryl ether catabolism. Purified LigG showed an approximately 300-fold higher specific activity for (βR)-GS-HPV than that for (βS)-GS-HPV, whereas purified LigQ showed an approximately six-fold higher specific activity for (βS)-GS-HPV than that for (βR)-GS-HPV. Analyses of mutants of ligG, ligQ, and both genes revealed that SYK-6 converted (βR)-GS-HPV using both LigG and LigQ, whereas only LigQ was involved in converting (βS)-GS-HPV. Furthermore, the disruption of both ligG and ligQ was observed to lead to the loss of the capability of SYK-6 to convert MPHPV. This suggests that GSH removal from GS-HPV catalyzed by LigG and LigQ, is essential for cellular GSH recycling during β-aryl ether catabolism.

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

Project description: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.

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