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 a cyclic monoterpene, CLPP. The structures of apo-CLDS and CLDS in complex with Tris, pyrophosphate, and Mg ion were refined at 2.00 Å resolution and 1.73 Å resolution, respectively. CLDS adopts a typical fold for cis-prenyl synthases and forms a homo-dimeric structure. An in vitro reaction using a regiospecifically H-substituted DMAPP substrate revealed the intramolecular proton transfer mechanism of the CLDS reaction. The CLDS structure and structure-based mutagenesis provide mechanistic insights into this unprecedented terpene synthase. The combination of structural and mechanistic insights advances the knowledge of intricate terpene synthase-catalyzed reactions.

“Head‐to‐Middle” and “Head‐to‐Tail” cis‐Prenyl Transferases: Structure of Isosesquilavandulyl Diphosphate Synthase

“…While this article was undergoing revision, another function/structure paper on a" head-to-middle" prenyltransferase,c yclolavandulyl diphosphate synthase,C LDS,w as reported. [16] Thestructure is very similar to what we find with Mcl22, although the mechanisms of action are different since…”

Structural and Mechanistic Insight into Terpene Synthases that Catalyze the Irregular Non‐Head‐to‐Tail Coupling of Prenyl Substrates

“…X‐ray crystallography analyses have revealed that the protein structures of UPP synthase (UPPS), LPPS, CLDS, and Mcl22 adopt a typical fold for the cis ‐type IDS family (Figure ). These structures have allowed mechanistic and structural comparison among the cis ‐type IDS homologous enzymes.…”

“…The intramolecular proton‐transfer mechanism of the CLDS reaction has been proposed on the basis of the combination of an in vitro reaction in which the regiospecifically 2 H‐substituted DMAPP substrate and the CLDS crystal structure are used (Figures and ) . The CLDS‐catalyzed reaction begins with ionization of the pyrophosphate leaving group of DMAPP with formation of the dimethylallyl cation in the S1 site.…”

“…[10] CLDS is the only terpene synthase that affords the c1'-2-4-3' coupling pattern (Figures 1 and 2). X-ray crystallography analyses have revealed that the protein structures of UPP synthase (UPPS), [11] LPPS, [12] CLDS, [13,14] and Mcl22 [15] adopt at ypical fold for the cis-type IDS family ( Figure 3). These structures have allowed mechanistic and structuralc omparison among the cis-type IDS homologous enzymes.…”

“…The intramolecular proton-transfer mechanism of the CLDS reactionh as been proposed on the basis of the combination of an in vitro reactioni nw hich the regiospecifically 2 H-substi-tuted DMAPP substrate and the CLDS crystal structure are used (Figures 2a nd 4). [13] The CLDS-catalyzed reaction begins with ionization of the pyrophosphate leaving group of DMAPP with formationofthe dimethylallyl cation in the S1 site. The reactive allylic carbocation subsequently undergoes nucleophilic attack by the double bond of the other DMAPPt of ormt he 1'-2 bond, and this yields at ertiary carbocation intermediate in the S2 site.…”