Crystal Structure Of Miltiradiene Synthase And Functional Characterization Of Sesquiterpene Synthase From Tripterygium Wilfordii

Terpenes constitute a large and structurally diverse family of natural molecules which display important biological activities and used for medical purposes.Terpenoids derives from several kinds of acyclic polyprenyl diphosphate substrates,farnesyl diphosphate(C15)and geranylgeranyl diphosphate(C20)are the linear precursor of sesquiterpene and diterpene,respectively.These linear polyprenyl substrates are cyclized by terpene synthases(TPSs)into products containing either a single ring or multiple rings,whereupon they are subjected to further modification by downstream enzymes.Abietane-related diterpenes,exhibit significant pharmacological activities including triptolide,tanshinone IIA and carnosic acid.Miltiradiene,abietadiene,levopimaradiene,and neoabietadiene are important skeleton precursors of abietane-related diterpene.Miltiradiene contains a characteristic double bond between C8 and C9.In plant,Miltiradiene is synthesized through two-step cyclization reaction which includes protonation-initiated cyclization of geranylgeranyl diphosphate(GGPP)to(+)-copalyl diphosphate(nor-CPP)by copalyl diphosphate synthase(CPS)and the ionization-initiated cyclization nor-CPP to miltiradiene by kaurene synthase-like(KSL).The relative area of miltiradiene synthase including conformation of nor-CPP and the specific double bond are very different from other reported diterpene synthases.Due to the lack of structural information,the recognition mode of substrate,as well as the catalytic processes and mechanism remain unclear.Cryptomeridiol,a typical eudesmane diol,is the active principle component of the antispasmodic Proximol.Although it has been used for many years,the biosynthesis pathway of cryptomeridiol has remained blur.The internal key enzymes for generating cryptomeridiol may include sesquiterpene cyclase and cytochromes P450 depending on the structure.Previous study have reported a novel sesquiterpenoid synthase from maize that can generate eudesmane-2,11-diol and minor cryptomeridiol.However,the isolation,functional characterization,mechanism of cryptomeridiol synthase are not well understood.Therefore,this study focused on two parts of work on the miltiradiene synthase and cryptomeridiol synthase.1 Structure and Mechanisms of Bifunctional Miltiradiene Synthase from Selaginella moellendorffiiWe determined three crystal structures of SmMDS from Selaginella moellendorffii in apo-state and GGPP-bound forms,representing 40 min and 4 h different states during its catalytic process,namely SmMDS,SmMDS40min,SmMDS4h.SmMDS adopts the classical plant diterpene synthases ??? modular.The first reaction that GGPP is converted to nor-CPP occurs in the class ? active site which locates in the cavity of ?? interface.The second reaction takes place in the a domain in which class ? reaction occurs and nor-CPP is converted to miltiradiene.In the apo-state SmMDS and ligand-binding state SmMDS40min and SmMDS4h structures,the class ? active pocket are similar and the pyrophosphate show slightly different conformation,while there exists a visible open-closed state in the class ? active site.The most significant change is the Trp381 side chain which is close to the tail of the modeled GGPP in class ? active site.Through mutagenesis studies,we demonstrated the key role of amino acids constituting the class ? active site.In addition,we identified an internal channel between the two active sites,which may facilitate the transfer of the nor-CPP from the class ? active site to the class ? active site.Furthermore,we identified a number of catalytic important residues in class ? active site and characterized several carbocation intermediate quenched side products through docking and mutagenesis studies.These results provide new insights into the catalysis mechanism of TPS and important reference for new diterpene modification and synthetic biology.2 cloning,functional characterization and synthetic biology application of cryptomeridiol synthase from Tripterygium wilfordii.Depending on the transcriptome of Tripterygium wilfordii,we cloned the full length of cDNA that encodes cryptomeridiol synthse(TwCS).The TwCS exhibited highest transcription level in the root bark of Tripterygium wilfordii by real-time fluorescent quantitative PCR.In eukaryotic system,TwCS directly produce eudesmane-type sesquiterpene diols,mainly cryptomeridiol.Using strategies namely overexpressing upstream genes,fusion proteins,down-regulating competitive pathways,and regulating transcription factors,the biosynthesis pathway of cryptomeridiol was re-constructed in Saccharomyces cerevisiae.The final high-yield strain TH9(BY4741 erg9::?-200-176 rox1::mut/pYX212-IDI+TwCS/p424-tHMG1)produced 19.73 mg/L of cryptomeridiol in shake flask culture.In addition,we found that the differential expression of the TwHMGS exhibited effect on terpenes biosynthesis in Tripterygium wilfordii.