| Summary: | <i>E. coli</i> thioesterase ‘TesA is an important enzyme in fatty acid production. Medium-chain fatty acids (MCFAs, C6-C10) are of great interest due to their similar physicochemical properties to petroleum-based oleo-chemicals. It has been shown that wild-type ‘TesA had better selectivity for long-chain acyl substrates (≥C16), while the two mutants ‘TesA<sup>E142D/Y145G</sup> and ‘TesA<sup>M141L/E142D/Y145G</sup> had better selectivity for medium-chain acyl substrates. However, it is difficult to obtain the selectivity mechanism of substrates for proteins by traditional experimental methods. In this study, in order to obtain more MCFAs, we analyzed the binding mode of proteins (‘TesA, ‘TesA<sup>E142D/Y145G</sup> and ‘TesA<sup>M141L/E142D/Y145G</sup>) and substrates (C16/C8-N-acetylcysteamine analogs, C16/C8-SNAC), the key residues and catalytic mechanisms through molecular docking, molecular dynamics simulations and the molecular mechanics Poisson–Boltzmann surface area (MM/PBSA). The results showed that several main residues related to catalysis, including Ser10, Asn73 and His157, had a strong hydrogen bond interaction with the substrates. The mutant region (Met141-Tyr146) and loop<sub>107–113</sub> were mainly dominated by Van der Waals contributions to the substrates. For C16-SNAC, except for ‘TesA<sup>M141L/E142D/Y145G</sup> with large conformational changes, there were strong interactions at both head and tail ends that distorted the substrate into a more favorable high-energy conformation for the catalytic reaction. For C8-SNAC, the head and tail found it difficult to bind to the enzyme at the same time due to insufficient chain length, which made the substrate binding sites more variable, so ‘TesA<sup>M141L/E142D/Y145G</sup> with better binding sites had the strongest activity, and ‘TesA had the weakest activity, conversely. In short, the matching substrate chain and binding pocket length are the key factors affecting selectivity. This will be helpful for the further improvement of thioesterases.
|
|---|