Exploration Of The Catalytic Function Of The Key Amino Acid Sites From The HheC Loop 3

Halohydrol dehalogenases are catalysts for the degradation of organic halides in nature,especially the halohydrol dehalogenase HheC,which catalyzes the synthesis of optically pure chiral epoxides of Short-chain o-halanols with high enantioselectivity for different aromatic or aliphatic compounds,and has high economic value in both environmental management and biopharmaceuticals.The active region of HheC consists of four loops,loop 1:D80-P88;loop 2:S132-L142;loop 3:P175-P188;and loop 4:M245-E254,loop 3 is the halogen ion binding region.Based on the results of previous loop 3 alanine scans in the lab,the loci 12,177,185,186,and 187 were found to be highly preferential for the aromatic amino acids Phe/Tyr.To investigate the specific effects of these loci on HheC catalysis and the synergistic effects of different mutant loci on enzymatic activity catalysis.In this experiment,a random combination mutation library was constructed for F12,Y177,Y185,F186,Y187,and each locus contained only F or Y mutations,with a total of 32 mutants.A 96-well plate was selected and screened with 1,3-DCP as the substrate for 6 rounds with a total of528 library accesses,yielding a total of 20 mutants.The results of the screening revealed that the dominant mutants all had the F12Y or F186Y mutation site,and the best performing mutant was F12Y/F186Y.The inferior mutants were those with Y177F and Y187F double mutations.Preliminary crude enzyme activity analysis was first performed on the mutants obtained from the screening to verify the enzyme activity trend of the screening results.Next,the mutants that showed better catalytic activity for the substrate 1,3-DCP were selected from the crude enzyme enzyme activity and assayed for the enzyme kinetic parameters.The k_cat/K_m of F12Y/F186Y was found to be the largest with the best enzyme activity and the smallest K_m value,indicating the greatest affinity for 1,3-DCP.k_cat value of Y177F/F186Y was the largest at 91.5 s^-1,which was 2.3 times higher than that of WT,indicating a larger increase in catalytic rate.Analysis of the protein structure of HheC and the results of MD simulations revealed that F12 and F186 are close to the catalytic triad,and when they mutated to tyrosine,narrow the substrate binding pocket.In the short halide ion release channel of HheC,the F186 site is present throughout,and when it is mutated to tyrosine,it brings the halide ion release channel as a whole closer to the catalytic triad,accelerating the release of halide ions.F12 is located at the head of the halide ion release channel,and when it is mutated to tyrosine,the increased hydroxyl group,not only provides a hydrophilic environment,but also increases the radius of the channel head,thus promoting the release of halide ions.For Y177,Y185,and Y187,there are more hydrogen bonds with surrounding amino acids by themselves,thus maintaining the halogen ion release channel.when mutated to phenylalanine,these hydrogen bonds will be lost,greatly affecting the rate of halogen ion release.when Y177,Y185,and Y187 are all mutated to phenylalanine,they are completely inactive for 1,3-DCP.The short channel is the main channel for halide ion release,and the regulation of halide ion release by tyrosine is more favorable in loop 3.