A novel labda-7,13e-dien-15-ol-producing bifunctional diterpene synthase from Selaginella moellendorffii.

Vascular plants invariably contain a class II diterpene cyclase (EC 5.5.1.x), as an ent-copalyl diphosphate synthase is required for gibberellin phytohormone biosynthesis. This has provided the basis for evolution of a functionally diverse enzymatic family.[1] These biocatalysts fold their substrate, the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP), to bring the terminal three carbon-carbon double bonds into proximity with each other, and then carry out bicyclization via a protonation-initiated carbocation cascade reaction. The resulting labda-15-en-8-yl+ diphosphate intermediate is most commonly quenched by deprotonation at an exocyclic methyl, as in the production of labdadienyl/copalyl diphosphate (Scheme 1). Alternatively, the bicyclized labda-15-en-8-yl+ diphosphate intermediate can be captured by water prior to deprotonation, to form hydroxylated compounds such as labda-15-en-8-ol diphosphate.[2] In addition, this intermediate can undergo subsequent rearrangement via 1,2-hydride and/or methyl shifts, starting with the hydrogen substituent on the neighboring endocyclic methine (C9).[3] However, terminating deprotonation at the neighboring endocyclic methylene (C7) has not previously been observed. Here we report that the lycophyte Selaginella moellendorffii contains a bifunctional diterpene synthase, SmCPSKSL1, which catalyzes just such a class II cyclization reaction. In particular, SmCPSKSL1 produces an endocyclic double bond isomer of copalyl diphosphate (CPP), as well as carries out subsequent replacement of the diphosphate by a hydroxyl group to form labda-7,13E-dien-15-ol. Although this is a known plant metabolite,[4] and a small family of bioactive derived natural products is known from a phylogenetically diverse group of plants,[4-5] its biosynthesis has not been previously investigated. Our results demonstrate that this diterpenoid can be generated by a single bifunctional diterpene synthase that directly generates the endocyclic double bond, as well as hydroxyl group. Scheme 1 Cyclization of GGPP to CPP. S. moellendorffii is a basal vascular land plant whose genome has been sequenced, and which contains a number of putative diterpene synthases.[6] At least one of these appears to be a bifunctional diterpene synthase (corresponding to the Joint Genome Institute gene accession Selmol 112927), as it contains both the DXDD motif associated with class II activity and DDXXD motif associated with allylic diphosphate ionization initiated class I activity.[7] In an attempt to determine the biochemical function of the encoded enzyme, a corresponding synthetic gene was obtained (Genscript), with codon-optimization for expression in Escherichia coli. Unfortunately, no diterpene synthase activity was observed with the resulting recombinant protein. The corresponding gene was then cloned from the lycophyte itself, revealing a few key differences from the predicted gene sequence, most notably a nine-nucleotide insertion. Expression of this native gene (SmCPSKSL1) in E. coli provided diterpene synthase activity. Specifically, upon co-expression with a GGPP synthase, using a previously described metabolic engineering system,[8] this bifunctional diterpene synthase predominantly led to production of an unrecognized diterpenoid by the recombinant bacteria, albeit with a few other minor products also observed (Figure 1). Figure 1 SmCPSKSL1 produces labda-7,13E-dien-15-ol (peak 1). A) GC-MS chromatogram of extract from E. coli engineered to co-express SmCPSKSL1 with a GGPP synthase. B) MS of peak 1 (labda-7,13E-dien-15-ol), with inset to clearly depict the molecular ion peak at ... The structure of this predominant diterpenoid, representing ~80% of the total product output, was determined by NMR. Sufficient amounts of material for this purpose was produced by increasing metabolic flux into isoprenoid/terpenoid metabolism in the metabolically engineered bacteria, as previously described,[9] and extraction from 2 L cultures of the resulting recombinant bacteria. Following purification via silica gel and reverse-phase high-performance liquid chromatography, 18 mg of this compound was obtained, enabling straightforward characterization. In particular, the structure was determined using HMBC, HSQC, and COSY data, leading to its assignment as labda-7,13E-dien-15-ol, which was confirmed by comparison to data in the literature.[4] The presence of the primary C15 hydroxyl group essentially represents hydrolysis of the allylic diphosphate ester bond. While similar hydroxyl replacement of the diphosphate has been observed with other class I terpene synthases,[7] here it was possible that this was a result of direct hydrolysis catalyzed by endogenous phosphatases in the E. coli based metabolic engineering system. To verify that this was catalyzed by SmCPSKSL1, the recombinant enzyme was expressed with a 6×His tag, purified, and shown to catalyze direct production of labda-7,13E-dien-15-ol in vitro, even in the presence of phosphatase inhibitor. Thus, SmCPSKSL1 is bifunctional, catalyzing both class II bicyclization of GGPP and class I ionization of the resulting intermediate with the addition of water to C15 prior to deprotonation (Scheme 2). SmCPSKSL1 then provides the first example of a class I diterpene synthase that catalyzes such direct replacement of the diphosphate with a hydroxyl, although others that incorporate water following cyclization are known.[10] In addition, the mass spectra of the minor products suggests that these may correspond to production of labdatriene(s) and labda-7,14-dien-13-ol(s) – e.g., the apparent molecular ion peaks at m/z = 272 or 290, respectively – albeit of unknown configuration. While these will be more precisely characterized in future studies, production of such diterpenes is further consistent with class I mediated ionization of the postulated labda-7,13E-dienyl diphosphate intermediate produced by the class II active site, as analogous compounds are produced by other class I diterpene cyclases.[3a, 11] Scheme 2 SmCPSKSL catalyzed production of labda-7,13E-dien-15-ol from GGPP. Labdanes can be produced as several different stereoisomers, which presumably result from folding of GGPP into alternative prochiral conformations prior to class II diterpene cyclase catalyzed bicyclization.[1] While we can not assign the absolute stereochemistry of the SmCPSKSL1 labda-7,13E-dien-15-ol product, the observed NOE between the hydrogen substituents on C20 and C11 demonstrates that these C10 and C9 substituents exhibit a cis configuration. This then implies a pro-chair-chair conformation of GGPP in the class II active site. Based on analogous chemical shifts relative to those observed with other labdane-related diterpenes, as well as that of the previously characterized corresponding natural product,[4] we tentatively assign the SmCPSKLS1 product to the normal absolute configuration. Regardless of absolute configuration, the production of an endocyclic double bond bicycle strongly indicates that SmCPSKSL1 catalyzes a unique class II reaction. Specifically, that the terminal labda-15-en-8-yl+ diphosphate intermediate is quenched by deprotonation at the neighboring C7 methylene. It is relatively easy to envision a concerted bicyclization reaction in the commonly catalyzed class II diterpene cyclase production of CPP, due to the evident ability to align the departing proton from the rotatable methyl group with the π orbitals of the relevant carbon-carbon double bonds (Scheme 1). Given the production of a C7 endocyclic double bond isomer observed here, the relative positioning of the C7 hydrogen substituents was examined. Notably, molecular modeling demonstrates that one of these hydrogens also can be closely aligned with the neighboring π orbital (e.g. C7α in the assumed “normal” configuration; Figure 2), enabling a similarly concerted bicyclization reaction, and providing insight into the conformation of the GGPP substrate in this class II active site. Figure 2 Relative configuration of C7 hydrogen substituents with π orbital of the sp3 hybridized C8 in SmCPSKSL1 catalyzed class II bicyclization. Depicted is putative moncyclic intermediate (R = allylic isoprenyl diphosphate “tail”), with ... In conclusion, characterization of a bifunctional diterpene synthase from S. moellendorffii has revealed novel enzymatic activity. Most notably, this SmCPSKSL1 defines a new class II reaction, which further enabled some insight into GGPP substrate conformation, as well as providing the first example of a class I diterpene synthase carrying out replacement of the diphosphate with a hydroxyl. In addition, given the uncertain origins of the endocyclic double bond, along with that of the hydroxyl group, the biosynthesis of labda-7,13E-dien-15-ol was previously unclear, and potentially required several enzymatic steps. The results reported here demonstrate that labda-7,13E-dien-15-ol can instead be produced by a single (albeit bifunctional) enzyme. This highlights such simple biosynthesis, only requiring changes in a single enzymatic gene, as a potential mechanism enabling the repeated convergent evolution that presumably then underlies the observed phylogenetically scattered production of the derived family of natural products.