Abstract

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.

Highlights

  • Lignin is one of the major components of plant cell walls, accounting for 15–40%1

  • We identified the GS-HPV-converting GST genes in SYK-6 and uncovered the roles of these genes in β-aryl ether catabolism, since the genes involved in GS-HPV catabolism have not been clarified in SYK-6 to date

  • High-performance liquid chromatography (HPLC) analysis of the reaction mixtures showed that MPHPV was converted into GS-HPV by purified LigF and LigE at 60 min (Fig. S2A–C)

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Summary

Introduction

Lignin is one of the major components of plant cell walls, accounting for 15–40%1. The industrial production of useful substances from lignin is desired, since it is the most abundant aromatic resource and the second most abundant bioresource on Earth after cellulose. LigF and LigE cleave the β-aryl ether linkage through an SN2 nucleophilic attack of GSH on the β-carbon of the s­ ubstrates[18,19] Another GST, LigG, catalyzes the cleavage of the thioether linkage in (βR)-GS-HPV by transferring the (βR)-GS-HPV GSH moiety to another GSH molecule, producing achiral β-hydroxypropiovanillone (HPV) and oxidized glutathione (GSSG)[14,16]. Kontur et al reported that N­ aGSTNu, belonging to the Nu-class GST, showed activity toward both GS-HPV isomers, and it was the only enzyme essential for converting both GS-HPV isomers in Novosphingobium aromaticivorans DSM ­1244424 They showed that the gene product of SLG_04120, a putative Nu-class GST in SYK-6, had GSH-removing activity for both GS-HPV isomers. We identified the GS-HPV-converting GST genes in SYK-6 and uncovered the roles of these genes in β-aryl ether catabolism, since the genes involved in GS-HPV catabolism have not been clarified in SYK-6 to date

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