Studies On The Selectivity Mechanism Of Wild-type Thioesterase ’tesA And Its Mutants For Medium-and Long-chain Acyl Substrates

E.coli thioesterase’Tes A,is the most widely used thioesterase of lacking a signal peptide in the synthesis of type II fatty acids in recent years.’Tes A can catalyze the hydrolysis of acyl carrier protein and releases free fatty acids.It is a key enzyme in fatty acid production.Medium-chain fatty acids（MCFAs,C6-C10）have received widespread attention due to their similar physicochemical properties to petroleum-based petrochemicals compared to long-chain fatty acids（LCFAs,≥C12）.Although much progress has been made in the synthesis of fatty acids by microorganisms,the amount of MCFAs with high application value is still low,and there is no effective way to produce medium-chain chemicals in industrial microorganisms.Therefore,how to increase the production of MCFAs by mutating some residues of E.coli thioesterase’Tes A to improve the selectivity for medium-chain acyl substrates is an important topic.Previous experiments have shown that the wild-type‘Tes A had better selectivity for long-chain acyl substrates,while the two mutants‘Tes A^E142D/Y145G and‘Tes A^{M141L/E142D/Y145G} had better selectivity for medium-chain acyl substrates.However,it was difficult to obtain the selectivity mechanism of substrates to proteins by traditional experimental methods.In this study,in order to get more MCFAs,we analyzed the binding mode,the key residues,catalytic mechanisms and the reasons for the different selectivity of different mutants(the wild-type‘Tes A,the two mutants‘Tes A^E142D/Y145G and‘Tes A^{M141L/E142D/Y145G})for acyl substrates of different chain length（C16/C8-N-acetylcysteamine analogues,C16/C8-SNAC）through molecular docking,molecular dynamics（MD）simulations and the molecular mechanics Poisson–Boltzmann surface area（MM/PBSA）.The results showed that there were four major regions of substrate binding to the enzyme in all complexes,including the oxygen anion hole（Asp9-Tyr15,Gly44-Thr46,Asn73-Gln80）,the catalytic triads（Ser10,Asp154,His157）,loop_107-113 and the mutant region（Met141-Tyr146）.The key residues associated with catalysis,including Ser10,Asn73 and His157,have strong interactions with substrates and exist mainly in the form of hydrogen bonds.The mutant region and loop_107-113 have some moderately strong interactions with the aliphatic chain tail of the substrate,mainly in the form of Van der Waals contribution.The reason for the difference in activity between different mutants of E.coli thioesterase for different substrates can be attributed to the different interactions of the key regions of the protease that bind to both ends of the polar head and non-polar tail for different chain length substrates.For the long-chain acyl substrate C16-SNAC,except for the triple mutant’Tes A^{M141L/E142D/Y145G},which has a large conformational change,’Tes A and’Tes A^E142D/Y145G can distort the C16-SNAC into a bigger high-energy conformation for the catalytic reaction due to their strong interactions at both head and tail ends.For the medium-chain acyl substrate C8-SNAC,the head and tail are difficult to bind to the protease at the same time due to the insufficient chain length,which makes the substrate binding position more variable.So the triple mutant with a better binding site has the strongest activity during the computational study,and conversely,the wild type has the weakest activity.In summary,the matching acyl chain length and binding pocket length are the key factors affecting substrate selectivity.This study provides a common approach to computational chemistry for researchers in the field of computation,and will also help to improve thioesterases in future scientific research.