Efficient Biosynthesis Of Heterodimeric C3-Aryl Pyrroloindoline Alkaloids

Pyrroloindoline alkaloids,which account for about 1%of the total number of natural alkaloids,are a large class of important active nature products.Among them,C3-aryl pyrroloindoline alkaloids always exhibit a broad array of biological properties.However,compared to the large number of homodimeric C3-aryl pyrrolidine alkaloids,only 5 heterodimeric products were reported.Up to now,there is no unified and efficient methods for constructing and expanding the heterodimeric molecular skeleton of this family,and as a result,this have severely limited the pharmacological and biological studies of heterodimeric C3-aryl pyrrolidine alkaloids.Therefore,in our study,we explored the biosynthesis,structure expansion and biological activity of heterodimeric C3-aryl pyrrolidine alkaloids-Naseseazines A/B/C,through four parts of research.The specific contents are as follows:1)Efficient biosynthesis of Naseseazines C and its analogues:Firstly,the key P450enzyme,Nasc B,was successfully identified by analyzing the biosynthetic pathway of Naseseazines C.Then,we demonstrated that Nasc B catalyzes a radical cascade reaction to form intramolecular and intermolecular carbon–carbon bonds with both regio-and stereo-specificity by means of Electron Paramagnetic Resonance(EPR),TEMPO inhibition experiments and DFT calculations.In addition,by constructing nasc B gene into a novel antilytic E.coli GB05dir-T7,we developed an efficient whole-cell catalytic system.By feeding 30 chemically synthesized cyclic dipeptide substrates,30 novel C3-aryl pyrrolidine alkaloids were successfully obtained.Finally,we found that some of these compounds have neuroprotective effects,especially compounds NAS-12,NAS-27,NAS-10 and NAS-11,which show better effects than positive control drug-nimodipine.2)Efficient biosynthesis of Naseseazines A/B and its analogues:Firstly,another key P450 enzyme responsible for the biosynthesis of Naseseazines A/B,Nasb B,was identified by heterologous expression and total chemical synthesis experiments.Then,in vitro reconstitution of Nasb B reaction confirmed its catalytic activity for the biosynthesis of Naseseazines A/B.Subsequently,by constructing Nasb B gene into Mycobacterium system,we developed another highly efficient whole-cell catalytic system,to which 30 chemically synthesized cyclic dipeptide substrates were fed,and fortunately another 10 novel Naseseazines A/B analogues were successfully obtained.In addition,through homology modeling and molecular docking methods,two key amino acids(V165 and F361)based on Nasb B were found out to play an important role in the regulation of Naseseazines B and Naseseazines C production;Finally,we also found that compound NAS-37 has modest neuroprotective function by bioactivity experiments.3)Creating structural varieties of NASs through genome mining:First,the P450enzymes with high identities compared to Nasc B were found in NCBI database,from which the P450 enzymes with CDPS or NRPS genes nearby were selected as the target P450 enzymes that may catalyze the formation of heterodimeric C3-aryl pyrrolidine alkaloids.Then,by analyzing the evolutionary tree,other eight proteins,namely SA-P450,AO-P450,S1868-P450,F5053-P450,SAC-P4501,SAC-P4502,41663-P450 and STDA-P450,were finally determined.These target P450 proteins were expressed and purified by E.coli or Mycobacterium expression system,and moreover,a series of in vitro biochemical experiments and whole-cell catalytic experiments were carried out to screen the substrate scope of these P450 enzymes by using the 30 chemically synthesized substrates.Up to now,15 newly dimeric products were found and the corresponding structures are currently being analyzed.4)Creating structural varieties of NASs through protein engineering:In this part,we firstly found that F5053-P450 can produce both Nasc B and S1868-P450 products when catalyzed the substrate c W_L-P_L,and then by analyzing the amino acid sequences of these three enzymes we noticed that the amino acid sequences in the first half part of F5053-P450 are identical with Nasc B,while the second half part is mostly identical with S1868-P450.These results suggest that there must be some amino acids which are important for regulating regioselectivity and stereoselectivity of reactions.Therefore,fragment substitution and single point mutation experiments were carried out and we successfully found that amino acid residues at 68,89,287 and 291 sites play a key role in regulating the regioselectivity and stereoselectivity of the catalytic reaction.After mutating the four sites into the site in S1868-P450 simultaneously in nasc B protein,the products of S1868-P450 become the main products.Then,76 mutants were obtained by saturated mutation of the four sites,and all of them were constructed into whole-cell catalytic system.Activity analysis using c W_L-P_L as substrate was carried out in order to discover new structural skeletons with different regions/stereoselectivity.And at present,the analysis work is in progress.In conclusion,a series of P450 enzymes with catalytic activity for production of heterodimeric C3-aryl pyrrolidine alkaloids have been successfully identified.Two simple and efficient whole-cell catalytic systems have been developed.By screening30 cyclodipeptide substrates,55 novel dimerization products have been found.Four amino acid sites(68,89,287,291)that play a key role in the regulation of reaction region/stereoselectivity were identified by genome mining and enzyme engineering strategies,and now saturated mutations at four sites were performed to further discover the new heterodimerization framework.