Improvement in the Thermal Stability of IsMHETase by Sequence and Structure-Guided Calculation

In the degradation of poly(ethylene terephthalate) (PET), mono(2-hydroxyethyl) terephthalate (MHET) hydrolase (IsMHETase) plays a crucial role in the complete degradation of PET. Although IsMHETase was discovered concurrently with IsPETase, its structural and functional properties are not well understood. To enhance the thermal stability of IsMHETase, we selected six homologous proteins that share the closest evolutionary relationship for structure-based protein rational design, all exhibiting over 60% amino acid sequence identity with IsMHETase. Using FireProt, PROSS, and Consensus analysis, we identified the key mutation sites of IsMHETase. Sequence and structural analyses indicate that, among these seven proteins, all amino acids within 5 Å of the substrate-binding site are identical, with the exception of Ser131 and Phe415. Additionally, the amino acids within a 4 Å range of the catalytic triad are nearly identical. Through integrated free energy calculations, phylogenetic tree analysis, sequence analysis, and conservation analysis, we have identified a variant with four key mutations (termed IsMHETase-M1: N156G, T159V, E110A, A493P) that exhibits improved thermal stability. The selection of mutations during the protein modification process often requires considerable time. Our predictions have established a foundation for the rational design of IsMHETase and its homologous proteins.