Structure and Mechanism of the Monoterpene Cyclolavandulyl Diphosphate Synthase that Catalyzes Consecutive Condensation and Cyclization

Abstract

We report the three-dimensional structure of cyclolavandulyl diphosphate (CLPP) synthase (CLDS), which consecutively catalyzes the condensation of two molecules of dimethylallyl diphosphate (DMAPP) followed by cyclization to form ac yclic monoterpene,C LPP.T he structures of apo-CLDS and CLDS in complex with Tris,p yrophosphate,a nd Mg 2+ ion were refined at 2.00 resolution and 1.73 resolution, respectively.C LDS adopts at ypical fold for cisprenyl synthases and forms ah omo-dimeric structure.A n in vitro reaction using ar egiospecifically 2 H-substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure-based mutagenesis providem echanistic insights into this unprecedented terpene synthase.T he combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase-catalyzed reactions.Terpenoids represent an important class of biologically active compounds owing to their structural diversity. [1,2] The diverse structures of terpenoids are derived from two simple 5-carbon units:i sopentenyl diphosphate (IPP) and DMAPP. An isoprenyl diphosphate synthase (IDS) catalyzes the canonical and sequential "head-to-tail" condensation of IPP to DMAPP to produce al onger acyclic polyprenyl diphosphate such as C 10 geranyl diphosphate (GPP) [eq. (1) in Scheme 1a]. Ther esulting acyclic polyprenyl diphosphate substrates can be cyclized by terpene cyclases into single-ring or multi-ring products,w hich can be further diversified by subsequent biosynthetic modifications. [3] Thus,i nt erpenoid biosynthesis,two independent enzymes,anIDS and acyclase, consecutively catalyze the condensation and cyclization reactions,r espectively,y ielding structurally diverse terpenoids.I ntriguingly,h owever, CLDS alone catalyzes the "head-to-middle" condensation of two molecules of Scheme 1. Terpene synthase reactions. a) eq. (1), condensation of IPP and DMAPP by GPP synthase;eq. (2), condensation of two molecules of DMAPP and subsequent cyclization by CLDS;e q. (3), condensation of two molecules of DMAPP by LPPS;e q. (4), condensation of DMAPP and GPP by Mcl22. b) Condensationo ftwo molecules of the deuterium-substituted DMAPP substrate (4)and subsequent cyclization catalyzed by CLDS. To simplify the figure, only key deuteriumatoms are shown in the intermediates, Int1, Int2 and Int3.

Decoding Catalysis by Terpene Synthases

“…Head to tail ("1,4") condensation is required to generate these regular precursors, which will be the focus of this review, although other types of condensation are possible. 1,18 Terpenoids form a large part of the chemical repertoire of many plants, being used in attack, defense, signaling, and to attract pollinators. 19 Similarly, it has long been recognized that microbes can make terpenoids.…”

“…Condensation of another IPP unit gives farnesyl pyrophosphate (C 15 , FPP), the precursor to sesquiterpenoids, and so on. Head to tail (“1,4”) condensation is required to generate these regular precursors, which will be the focus of this review, although other types of condensation are possible. , …”

“…Head to tail (“1,4”) condensation is required to generate these regular precursors, which will be the focus of this review, although other types of condensation are possible. 1 , 18 …”