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 (<i>Is</i>MHETase) plays a crucial role in the complete degradation of PET. Although <i>Is</i>MHETase was discovered concurrently with <i>Is</i>PETase, its structural and functional properties are not well understood. To enhance the thermal stability of <i>Is</i>MHETase, 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 <i>Is</i>MHETase. Using FireProt, PROSS, and Consensus analysis, we identified the key mutation sites of <i>Is</i>MHETase. 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 <i>Is</i>MHETase-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 <i>Is</i>MHETase and its homologous proteins.