The Mechanism Of High Catalytic Efficiency Of Decarboxylase In Emodin Biosynthetic Pathway

Objective:Emodin is considered as a valuable pharmaceutical compound,which has diuretic,anti-inflammatory and antiviral bioactivities.Since the extraction of emodin in plants is time-consuming and costly.With the development of synthetic biology,heterologous synthesis of emodin had been becoming a possible alternative in engineered microorganisms.Decarboxylation catalyzed atrochrysone carboxylic acid(ACA)to generate emodin as the final product,which was the key final step in the emodin biosynthetic pathway.Therefore,in this work,firstly,according to the emodin biosynthetic pathway,the strain producing ACA was constructed in S.cerevisiae BJ5464-NpgA.Then,decarboxylase from different species was screened,researched the activities,and explored the efficient catalytic mechanism of decarboxylase through site-directed mutation.For further understanding the relationship between the structure and catalytic efficiency of decarboxylase,established a highly reliable model for the structure of enzyme protein and substrate binding with combining experimental data with molecular biology,which provided a certain reference for the decarboxylase modification.Method:1 The construction of the strain producing ACAThe key enzymes in the emodin biosynthetic pathway were genetically integrated by Cre/loxP into the genome of S.cerevisiae BJ5464-NpgA,which was stably produced ACA.2 Screen 8 significant amino acids.The decarboxylase was screened by sequence alignment from Group V NR-PKS gene cluster.The conversion rate of decarboxylase was detected,and screened the highest one.In order to exploring the efficient catalytic mechanism of the decarboxylase,we had predicted a series of non-conservatively important amino acid sites which potentially affected catalytic efficiency by sequence alignment,and performed site-directed mutations for its mechanism.3 The molecular simulation of decarboxylaseThrough I-TASSER,performed the homology modeling of the decarboxylase with the highest conversion rate,and predicted the three-dimensional structure of the decarboxylase.Drawing the three-dimensional structure of ACA by GuassView and docking molecularly between the decarboxylase and ACA for the efficient catalytic mechanism of the decarboxylase by molecular simulation through Autodock.Result:1 Constructed the strain that produced ACAIn the emodin biosynthetic pathway,introduced the key enzymes which were Acetyl-CoA Carboxylase,polyketide synthase,and metallo-β-lactamase-type-thioesterase into the gene expression vector which were pXP418 and pCEV-G4-Km.The key enzymes ACC1,SlA CAS,and HyTE were integrated into the S.cerevisiae BJ5464-NpgA genome by Cre/loxP,and constructed the strain sLY2(BJ5464-ACC1**-SIACAS-HyTE)which produced stably ACA.2 Screened 8 significant amino acids.Compared the catalytic efficiency of decarboxylase genes,named HyDC,TpcK,AfDC,PsDC,TaDC,PaDC,and ThDC,the decarboxylase HyDC had the highest conversion rate and the PsDC had the lowest one.Based on site-directed mutation of amino acids,explored eight important amino acids affecting the catalytic mechanism of decarboxylase,which were Lys7,Arg10,Ala61,Arg63,Ala67,Glu68,Ser72,and Glu89.Among the amino acids affecting the efficient catalytic mechanism of decarboxylase,Arg63 had the most influence,however,Lys7,Arg10,Ala61,Ala67,Glu68,Ser72,and Glu89 had small influence relatively.3 Decarboxylase molecular simulationCombined the data of site-directed mutagenesis,taken 3hfkB as a template,predicted the three-dimensional molecular structure of decarboxylase by homology modeling,we established a high reliable structure model of the protein and substrate binding,which analyzed the function of important amino acids.The amino acids Lys7,Arg10 and Glu89 gathered together to form the entrance of ACA,and Lys7 and Arg 10 was in the alpha helix,Glu89 was at the corner of the alpha helix and beta fold,and all were in the protein backbone structure for stabilizing the entrance of the emodin precursor.The amino acids Ala67,Glu68,and Ser72 were clustered together to show a significant positive charged region,formed an electrostatic attraction with the emodin precursor,and stabilized the binding of the decarboxylase and ACA.The amino acid Arg63 was formed hydrogen bonds which had a direct catalytic effect on the decarboxylation process.Conclusion:This study aimed at the emodin biosynthetic pathway and explored eight important amino acids that affected the catalytic mechanism of decarboxylase,and Arg63 was the key amino acid,which not only enriched the decarboxylase relationship between the structure and function,but also provided the values of reference for further enzyme modification.