Catalytic Mechanism of Aryl-Ether Bond Cleavage in Lignin by LigF and LigG

Given the abundance of lignin in nature, multiple enzyme systems have been discovered to cleave the beta-O-4 bonds, the most prevalent intermonomer linkage. In particular, stereo specific cleavage of lignin oligomers by glutathione S-transferases (GSTs) has been reported in several sphingomonads. Here, we apply quantum mechanics/molecular mechanics simulations to study the mechanism of two glutathione-dependent enzymes in the beta-aryl ether catabolic pathway of Sphingomonas sp. SYK-6, namely, LigF, a beta-etherase, and LigG, a lyase. For LigF, the free-energy landscape supports a S(N)2 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(N)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 beta-aryl ether cleavage pathway and reveals key structural features underpinning the ligninolytic activity detected in several sphingomonad GSTs.