A key finding to understand lignin biodegradation was the ability of lignin peroxidase (LiP) and versatile peroxidase (VP) to delocalize one oxidation equivalent from the buried heme to a surface tryptophan residue. In this way, the resulting radical is able to interact with the bulky lignin polymer, and transfer electrons to heme using a long-range pathway. The catalytic nature of the surface tryptophan had been demonstrated by directed mutagenesis using simple lignin models, but similar steady-state studies cannot be performed with lignin due to difficulties to estimate initial oxidation rates. However, it is possible to follow electron transfer from soluble (sulfonated) lignins from the “peroxidase side” (i.e. by estimating the constants for enzyme reduction under stopped-flow conditions) as shown in this study. Moreover, the possibility to reconstruct ancestral proteins permits nowadays to directly identify the evolutionary change responsible for every new enzyme property. Here, four LiP ancestors (red path in peroxidase main clade D, Figure left) were resurrected (by sequence reconstruction, heterologous expression in Escherichia coli, and in vitro activation) and their ability to transfer electrons to lignin were evaluated together with extant Phanerochaete chrysosporium LiP (PC-LiPA). As shown in the Figure (right) a relationship exists between the rate-limiting constant in lignin reactions (k3app from above stopped-flow analyses) and the presence of the catalytic tryptophan in AVPd, ALiP and PC-LiPA (absent from the previous CaPo and CaD ancestors). [1] This correlation was stronger for hardwood lignosulfonate, suggesting that the surface tryptophan was introduced in fungal peroxidases to better act on the more complex lignin developed by angiosperms. The appearance of a surface tryptophan paralleling the increase of peroxidase activity on lignin provides evolutionary evidence on the role of this residue in ligninolysis.

Resúmen de la comunicación oral presentada en 7th LignoBiotech international conference . Toulouse 2024 14-17 oct. 2024

Peer reviewed