Protein Structure And Catalytic Mechanism Of Alcohol Dehydrogenase 1 From Artemisia Annua L.

Malaria is a serious infectious disease mainly caused by Plasmodium falciparum and Plasmodium vivax.In 2020,approximately 241 million malaria cases were reported globally,among which about 627,000 died from that disease,with children under the age of 5 accounting for 77%of all malaria deaths.Artemisinin-combination therapies(ACTs)are the most effective clinical treatment methods recommended by the World Health Organization(WHO).However,the plant extraction of artemisinin could be affected by seasons and regions,resulting in unstable yields,which cannot meet its commercial needs.The development of synthetic biology has provided new ideas for the biosynthesis of natural drugs.Keasling’s group designed and integrated the biosynthetic pathway of artemisinic acid in engineered yeast cells.The production of artemisinic acid in Saccharomyces cerevisiae can reach 25 g L-1,and finally obtain artemisinin using a chemical semi-synthesis method.The key synthesis elements in the biosynthetic pathway of artemisinic acid were explored,and it was found that the key steps affecting the final synthesis of artemisinic acid are the redox reactions of the last two steps,in which the incorporation of Artemisia annua alcohol dehydrogenase 1(AaADH1)gene into the engineered yeast strain led to an 80%increase of artemisinic acid production,indicating that AaADHl plays an important role in the biosynthesis of artemisinic acid.In this paper,the protein structures and catalytic mechanism of AaADH1 were studied.In this study,AaADH1 was heterologously expressed by E.coli expression system,purified by Co2+-NTA affinity chromatography and anion exchange chromatography,and the soluble protein with a purity of more than 90%was obtained.The GC-MS method was used to detect the protein activity.The hanging-drop method was used for crystallization of apo AaADH1 and AaADH1-NAD+complex.After the primary screening and crystallization conditions optimization,diffraction data of the crystals were obtained at resolutions of 2.95 A and 1.80 A,respectively.The three-dimensional structures of apo AaADH1(PDB:7CYI)and AaADH1-NAD+complex(PDB:6LJH)were successfully resolved,and detailed structural analysis of the overall folding,coordination of zinc,and potential substrate binding sites was performed.Based on the structure of AaADH1-NAD+complex,artemisinic alcohol(ART)was docked into the active pocket of the binary structure,and the AaADH1-NAD+-ART ternary complex structure was constructed and optimized by molecular dynamics simulations.The stable conformation was selected for QM/MM MD(Quantum Mechanical/Molecular Mechanical Molecular Dynamics)to simulate its dehydrogenation reaction mechanism,focusing on the study of the coordination of zinc and the detailed process of hydride and proton transfer,and rationality of the inferred mechanism was verified the by comparing the calculated and experimental values of activation free energy and determination of the activities of key amino acid mutants.The results show that the coordination of substrate alcohols with zinc is not a necessary step in the mechanism,and the transfer of hydride and proton proceed in a"quasi-concerted" manner,which is determined by the unique allyl alcohol-like chemical structure of ART,which enriches the present molecular mechanisms of the reactions catalyzed by alcohol dehydrogenases.In this paper,the semi-rational design of key amino acids in the substrate binding cavity and substrate channel of AaADH1 was carried out,and a "small and refined" mutant library was constructed by using point saturation mutation technology.The mutant L366A with improved activity and solubility was screened with CCK8 method.In conclusion,this study successfully analyzed the three-dimensional protein structures of apo AaADH1 and AaADHl-NAD+binary complex,and explained the molecular mechanism of AaADH1 catalyzing ART to artemisinic aldehyde by QM/MM MD simulations.Notably,AaADH1 follows an unconventional and specific catalytic mechanism in which proton and hydride transfer proceed in a "quasi-concerted" manner.This study broadens the understanding of the catalytic mechanisms of alcohol dehydrogenases,further understands the biosynthetic pathway of artemisinin,and also sheds light on the rational design of artemisinin biosynthetic pathways to increase the production of artemisinin to meet the supply needs of malaria patients.