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. 2014 Jul:103:13-21.
doi: 10.1016/j.phytochem.2014.04.005. Epub 2014 May 5.

Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization

Alana J Jackson  1 , David M Hershey  1 , Taylor Chesnut  1 , Meimei Xu  1 , Reuben J Peters  2
Affiliations

Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization

Alana J Jackson et al. Phytochemistry. 2014 Jul.

Abstract

It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, a synthetic biology approach was applied to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a "splicing" error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, it is possible to assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.

Keywords: Diterpenoids; Natural products biosynthesis; Terpene synthases.

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Figures

Figure 1
Figure 1
Cyclization mechanisms for the diterpene synthase activities previously identified in castor bean by assays with cell-free extracts (Robinson and West, 1970b; Spickett et al., 1994), and their relationship to the classical mechanism for production of ent-kaurene (1). This classic mechanism proceeds via ionization of the allylic diphosphate ester bond in ent-CPP to trigger initial cyclization to the depicted ent-pimarenyl+ intermediate, followed by secondary cyclization to the depicted ent-beyeranyl+ intermediate that rearranges via the depicted ent-trachylobanyl+ intermediate en route to the ent-kauranyl+ intermediate that is quenched by deprotonation to yield ent-kaurene. As shown, the production of ent-beyerene (4) and ent-trachylobane (2) similarly arises from deprotonation of the corresponding carbocations.
Figure 2
Figure 2
Identification of products formed by the RcKS(L)s by comparison to authentic standards by GC-MS. Chromatographs of the products formed from ent-CPP by corrected RcKS1, RcKSL2, RcKSL3 and corrected RcKSL4, as indicated (with corrections as described in the text). Peaks are labeled with numbering corresponding to that used in the text (with structures shown in Figures 1 and/or 5), along with ent-labdatriene (6) and ent-pimaradiene (7). Also shown are mass spectra of the major enzymatic products, their retention times (RT) were 16. 22 min. for RcKSL1; 15.84 min. for RcKSL2; 15.67 min. for RcKSL3; and 15.46 min. for RcKSL4, along with those from the corresponding authentic standards [RT = 16.23 min. for ent-kaurene (1); 15.85 min. for ent-trachylobane (2); 15.67 min. for ent-sandaracopimaradiene (3); and 15.47 min. for ent-beyerene (4)].
Figure 3
Figure 3
Amino acid sequence alignment of the corrected RcKS1, RcKSL2, RcKSL3 and corrected RcKSL4 (corrections as described in the text). The primary DDxxD and secondary (N/D)Dxx(S/T)xxxE divalent magnesium ion binding motifs are underlined, with an asterisk (*) underneath the previously identified single residue switch position controlling progression to the secondary cyclization step of the KS reaction mechanism.
Figure 4
Figure 4
Phylogenetic tree for the biochemically characterized members of the TPS-e family. Constructed using MEGA5 (Tamura et al., 2011), specifically the Maximum-Likelihood method from codon-based alignment of the relevant open reading frames as described in the Experimental section. The gymnosperm derived PgKS and non-vascular plant derived PpCPSKS and JsCPSKS serve as the outgroup rooting the tree.
Figure 5
Figure 5
Cyclization mechanism derived from quantum chemical calculation (QCC) for the formation of ent-kaurene (1) and related diterpenes arising from secondary cyclization of the ent-pimarenyl+ intermediate formed by initial cyclization of ent-CPP. The QCC mechanism postulates concerted formation of ent-kaurenyl+, which then serves as a central intermediate en route to all other tetracyclic diterpenes, as well as the pentacyclic ent-trachylobane (2), with the corresponding ent-trachylobanyl+ intermediate not serving as a transition state (i.e., as has been suggested in the classic mechanism – see insert), but rather as arising independently.
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