Key Active-site Mining And Catalytic Mechanism Analysis Of Pentalenene Synthase

Pentalenene is an angular triquinane（containing three fused five-membered rings）sesquiterpene produced by a variety of Streptomyces species.Pentalenene is the precursor of antibiotic pentalenolactone,and can be used as a jet fuel blending agent.Pentalenene is synthesized by the ionization and cyclization of farnesyl pyrophosphate（FPP）catalyzed by pentalenene synthase（Pent S）,but the specific cyclization cascade reactions are not yet fully understood.To better understand the cyclization reactions,it is necessary to identify more key sites and elucidate their roles in terms of catalytic activity and product specificity control.In this study,we used pentalenene synthase from Streptomyces sp.PSKA01 as the target,performed a basic bioinformatics analysis,and established pentalenene synthetic pathway in E.coli BL21（DE3）to verify the Pent S catalytic function.Subsequently,semi-rational design and irrational design was used to modify Pent S,studying the effect of some amino acids on the enzyme activity.Molecular docking and molecular dynamics simulation were used to further analyze how the key amino acid affect the enzyme activity and product specificity.The main findings are as follows:（1）Basic bioinformatics analysis showed that the open reading frame of Pent S gene was 1014 bp,encoding 337 amino acids.Pent S with molecular weight of 37.9 k Da contains ⁸⁰DD××D/E,¹⁷³R,²¹⁹NSE/DTE and ³¹⁴RY conserved motifs,and is a barrel-shaped Class I terpene synthase containing of 11αhelices.The pentalenene synthetic pathway was constructed in E.coli BL21（DE3）,and the in vivo fermentation results showed that the main product was 98.3%pentalenene,the by-products were 0.3%thujopsene-I3,0.7%β-elemene and 0.7%two unknown sesquiterpenes（m/z 204）.The yield of pentalenene was up to 2.13 g/L.（2）Pent S was modified based on semi-rational design to investigate the effect of key amino acids on the catalytic activity of Pent S.Five mutation sites,T176A,T176G,I177G,A211T and A297S,were designed by homologous sequence alignment and three-dimensional structure analysis.Fermentation results showed that these five variants were important for the catalytic activity of the enzyme,and their mutations all resulted in different degrees of reduction or loss of catalytic activity.（3）Random mutant library and screening system was constructed to obtain a Pent S-13 mutant with significantly altered product specificity.Through sequencing and single-site mutant construction and functional verification,T182A,located on the G2 helix,was responsible for the phenotype of Pent S-13.The T182 saturation mutants were constructed,and the protein solubility of variants were examined,it was found that the T182 site could greatly affect the protein solubility.Subsequently,variants with the same solubility as the wild type were selected for fermentation verification,and the results showed that the catalytic activity of variants were completely lost when threonine was mutated to amino acids with larger side chains（phenylalanine,lysine,leucine,methionine）;the relative activity of T182I mutant was less than 3%and the product distribution was significantly changed;the T182A,T182C,T182S and T182V variants still retained most of their catalytic activity,with the T182A,T182C and T182V variants showing a significant change in product partitioning.（4）Molecular docking and molecular dynamics simulations were used to analyze the changes in the active cavity and FPP conformation of wild type and variants（T182A,T182C,T182S,T182V,T182I,T182F,T182K,T182L and T182M）of Pent S.The results showed that the T182 key active-site caused a significant change in the conformation of the FPP by affecting the volume and shape of the active cavity,and disturbing hydrophobicity/polarity interactions,which affected the stability of substrate binding and leaded to changes in the yield of pentalenene.Based on trajectory dynamical phase clustering calculations,the relationship between the occupancy of different relaxation conformations of FPP and the proportion of different products in wild type and variant systems was systematically analyzed.The results showed that the conformational changes of FPP also affected the possibility and frequency of attack on C1 cationic intermediates by other carbon atoms,increasing the formation of different carboncation intermediates,which in turn leaded to changes in product diversity.These results demonstrated the important role of the T182 site on the binding activity and regioselectivity of Pent S.The T182 residue identified in this study was not previously reported,and this key active site had an important role in the activity and product distribution of pentalenene synthase.Based on the results and analytical methods of this study,which contributed to understand the relationship between the structure and function of terpene synthase,laid the foundation for further analysis of the stereoselective mechanism of terpene synthase,improved the catalytic efficiency and product specificity of the enzyme,and provided theoretical references for the engineering of other terpene synthases.