Structure-Guided Ancestral Evolution of HmfF Yields a Robust Decarboxylase for Terephthalic Acid Degradation

Enzymatic conversion of poly(ethylene terephthalate) into terephthalic acid (TPA) represents a major frontier in environmental biotechnology, yet downstream TPA biotransformation remains constrained by dioxygenases, and no natural TPA decarboxylase has been reported. Here, we combine ancestral sequence reconstruction with structure-guided evolution to engineer HmfF, a 2,5-furandicarboxylic acid decarboxylase, yielding TPA decarboxylase activity. Guided by phylogenetic and structural analyses, 17 ancestral variants were reconstructed, among which HmfF_N1 exhibits exceptional thermostability with T m over 86 °C and enhances prenylated flavin mononucleotide (prFMN) cofactor binding. Substrate profiling across furandicarboxylic acids, terephthalic acid, and hydroxybenzoic acids shows that HmfF_N1 catalyzes decarboxylation with superior efficiency toward TPA. Relative to the reported enzyme PtHmfF from Pelotomaculum thermopropionicum, HmfF_N1 harbors 42 distal substitutions, and molecular dynamics simulations reveal that HmfF_N1 stabilizes both the prFMN cofactor and the TPA substrate. This study demonstrates that integrating structural insights into ancestral evolution enables the generation of robust enzymes, providing a promising biocatalyst for sustainable plastic utilization.