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

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 <i>Novosphingobium aromaticivorans</i> 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 <i>S</i>-transferase NaGST_Nu is the only enzyme needed to remove glutathione from both (<i>R</i>)- and (<i>S</i>)-GS-HPV in <i>N. aromaticivorans</i> We solved the crystal structure of NaGST_Nu 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 <i>Sphingobium</i> sp. SYK-6 (which can also break the β-aryl ether bond) and <i>Escherichia coli</i> (which cannot break the β-aryl ether bond) can also cleave (<i>R</i>)- and (<i>S</i>)-GS-HPV, suggesting that glutathione lyase activity may be common throughout this widespread but largely uncharacterized class of glutathione <i>S</i>-transferases.