Function,Mechanism And Crystallographic Study Of Sesquiterpene Synthase In Jungermannia Exsertifolia

Bryophytes can produce a variety of sesquiterpene compounds with biological activity and application value.The key to using synthetic biology methods to obtain sesquiterpene compounds more efficiently is the sesquiterpene synthase.In the previous research,the sesquiterpene synthase gene MTK was screened from the transcriptome analysis of Jungermannia exsertifolia,which are rich of sesquiterpene compounds.In this study,we conducted bioinformatics analysis,functional identification,protein crystallization and catalytic mechanism research on MTK..According to the bioinformatics analysis,the ORF of MTK gene is composed of1068 bp,encoding 355 amino acids.The predicted MTK protein molecular weight is40.4 k Da.It has two conserved metal binding motifs DDxxx and NSE/DTE and belongs to Microbial terpene synthase-like terpene synthase.The recombinant strain WQ011 p RS426-MTK was constructed.The fermention products were 35.17%?-Chamigrene,24.81% ?-Himachalene,19.89% Herolidol,7.97% ?-Chamigrene,7.82%(-)-Thujopsene and 4.35%(+)-?-Longipinene.The recombinant strain E.coli BL21(DE3)p ET28a-MTK was constructed.The optimal induction condition for inducing protein is 0.5 m M IPTG for 16?.Through multi-stage purification,MTK protein with higher purity was obtained and protein crystallization experiments were conducted.The optimal condition for protein crystallization by oil drop method is as follow: the protein concentration is 7 mg/m L,adding 0.5-0.7 ?L of PEG/ION-36,no additives and for 16?.The three-dimensional structure of the complex of MTK protein and substrate was constructed by homology modeling.18 amino acids constituted the active pocket of the protein.His71,Asp106,Phe179,Thr214,Asn256 and Tyr337 were selected for the mutation study and six mutant strains WQ011 p RS426-MTK H71 P,WQ011 p RS426-MTK D106 E,WQ011 p RS426-MTK F179 L,WQ011 p RS426-MTK T214 S,WQ011 p RS426-MTK N256 D and WQ011 p RS426-MTK Y337 F were obtained.According to the fermentation results,it can be seen that Asp106 and Asn256 on the conserved sequence of MTK protein are very important for the catalytic activity of the enzyme,His71 plays an important role in the cyclization reaction,Phe179 supports the active pocket,Thr214 slightly affects the C1-C6 cyclization reaction,Tyr337 participates in the proton transfer reaction system,which has an important effect on the departure of the phosphate group.This study preliminary explored the function and structure of sesquiterpene synthase MTK,and the analysis of its catalytic mechanism,which provided a theoretical basis for the future research of the Microbial terpene synthase-like terpene synthase.