Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the ?-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereospecific cleavage of lignin oligomers by glutathione S-transferases (GSTs) has been reported in several sphingomonads. Here in this paper, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the ?-aryl ether catabolic pathway of <
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Sphingomonas sp.<
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SYK-6, namely, LigF, a ?-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a SN2 reaction mechanism, with the monoaromatic leaving group being promptly neutralized upon release. Specific interactions with conserved residues are responsible for stereoselectivity and for activation of the cofactor as a nucleophile. A glutathione conjugate is also released by LigF and serves the substrate of LigG, undergoing a S<
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N<
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2-like reaction, in which Cys15 acts as the nucleophile, to yield the second monoaromatic product. The simulations suggest that the electron-donating substituent at the para-position found in lignin-derived aromatics and the interaction with Tyr217 are essential for reactivity in LigG. Overall, this work deepens the understanding of the stereospecific enzymatic mechanisms in the ?-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.