All embryophyta produce the diterpene ent-kaur-16-ene (1). In vascular plants (i.e., tracheophytes) 1 serves as an intermediate in biosynthesis of the gibberellin phytohormones (Hedden and Thomas, 2012). Even in earlier diverging bryophytes 1 seems to serve as a precursor to an as yet undefined signaling molecule (Anterola et al., 2009; Hayashi et al., 2010; Miyazaki et al., 2014, 2015). Production of 1 from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP) proceeds via two distinct bicyclization reactions (Peters, 2010). The first is catalyzed by copalyl diphosphate synthases (CPSs) that are representative of class II diterpene cyclases and produce ent-CPP (2). 2 is then subsequently further cyclized and rearranged by KSs that are representative of class I (di)terpene synthases (Zi et al., 2014). While the reactions catalyzed by CPSs and KSs are mechanistically distinct and carried out in distinct active sites, in plants these enzymes seem to be derived from fusion of the genes for each enzyme – i.e., CPS and KS – potentially from bacteria (Morrone et al., 2009; Gao et al., 2012). However, the presumably bifunctional nature of the initially resulting enzyme appears to have been retained only in certain bryophytes (Hayashi et al., 2006; Kawaide et al., 2011; Kumar et al., 2016). The production of 1 in most plants appears to depend on separate CPS and KS that arose from duplication and sub-functionalization of the ancestral fused bifunctional enzyme, with retention of the original multi-domain structure (Zi et al., 2014). In turn, the CPS and KS required for the production of signaling molecules derived from 1, such as the gibberellins, seem to have given rise to closely related enzymes that mediate more specialized labdane-related diterpenoid metabolism via gene duplication and neo-functionalization (Zi et al., 2014). These enzymes then catalyze similar reactions, but yield different products. For example, many angiosperms contain small families of KS-like diterpene synthases (KSLs) that retain the multi-domain structure of KSs, but do not produce 1. These catalyze ionization of the allylic diphosphate ester bond that characterizes class I terpene synthases, and may even still react with 2. However, these enzymes then mediate distinct product outcome by either abbreviation of the KS reaction, or by mediating formation of a distinct series of carbocationic intermediates. These KSLs terminate catalysis via deprotonation, either directly or following the addition of water, leading to a variety of products (see Scheme 1 for examples).

The evolutionary scenario presented above implies that the ancestral enzyme produced ent-kaurene, with conservation of this function in all extant KSs. However, such conservation of function versus parallel evolution from related but functionally divergent ancestors is difficult to discern from phylogenetic analyses given the extended timescale over which land plants have evolved (Kumar et al., 2016). For example, while KSs contain highly conserved DDxxD and NDxx(G/S/T)xxxE amino acid sequence motifs, these are found in class I terpene synthases more generally. Specifically, because they are involved in binding magnesium divalent ion co-factors required for the initiating ionization of the allylic diphosphate ester bond (Christianson, 2006). On the other hand, KSs have been reported to contain a conserved isoleucine that plays a surprisingly critical role in promoting the complex bicyclization and rearrangement reaction catalyzed by these enzymes (Scheme 1). In particular, substitution of threonine for this Ile leads to an abbreviated reaction, only cyclization of the ent-CPP substrate to ent-pimara-8(14),15-diene (3), representing deprotonation of the ent-pimar-15-en-8-yl carbocation formed by initial cyclization (Xu et al., 2007b). This effect has been interpreted as resulting from electrostatic stabilization of this carbocationic intermediate by the hydroxyl of the introduced Thr side-chain, rather than the hydroxyl group acting as a general base. The presence of the inert Ile hydrocarbon side-chain then allows carbocation migration toward the diphosphate anion co-product via further cyclization and ring rearrangement prior to terminating deprotonation (Zhou and Peters, 2011). However, conservation of this Ile and the effect of Thr substitution was originally only reported for KSs from angiosperms (Xu et al., 2007b). This Ile is conserved in a KS from gymnosperms (Keeling et al., 2010), with a similar effect on product outcome observed upon substitution with alanine as well (Zerbe et al., 2012). Thus, this Ile seems to be conserved in KSs throughout seed plants (i.e., spermatophyta). However, the identity and importance of this residue has not yet been investigated in KSs from even earlier diverging plants. Moreover, the similar effect of Thr or Ala substitution on product outcome also leaves some question as to the mechanistic role of the residue at this position given that Ala would be expected to be as inert as Ile. Here the conservation of this Ile and effect of Thr substitution is extended to KSs from the earliest diverging plants (i.e., bryophytes), including an example of a bifunctional CPS/KS, along with additional mutational analysis of this critical residue that resolve its role in the enzymatically catalyzed reaction.

The results reported here not only demonstrate conservation of the unique importance of a key Ile in the complex cyclization and rearrangement reaction catalyzed by plant KSs throughout the embryophyta, but also further extend our understanding of the underlying mechanism. For example, the stereospecific addition of water to generate the 8α-hydroxy-ent-pimar-15-ene product (7) observed upon substitution of the relevant I638 in AtKS indicates the orientation of this residue relative to the ent-pimar-15-en-8-yl⁺ intermediate in the catalyzed reaction. Specifically, in order for this to occur, the water must attack from ‘underneath’ the ring structure (Scheme 2). This presumably further defines the relative positioning of the key Ile residue, as it is changes of this amino acid side chain that enable such access.

Although the observed effects might suggest the hypothesis that these substitutions enable water to bind and act as a general base that directly deprotonates the ent-pimar-15-en-8-yl⁺ intermediate, this seems somewhat unlikely given previous results reported for class II diterpene cyclases that also catalyze reactions with similar carbocationic intermediates. In particular, while there is strong evidence that a specific water acts as the general base in the ent-CPP synthase from A. thaliana, this water is tightly constrained by several interactions with the enzyme. This includes the side chains of a histidine and asparagine, substitution of either of which with Ala is sufficient to lead to predominant formation of a hydroxylated product (Potter et al., 2014). Moreover, simply opening up space to create a polar ‘pocket’ at the site of the catalytic base in the class II diterpene cyclase active site of the abietadiene synthase from Abies grandis (i.e., either substitution of Ala or aspartate for histidine, or phenylalanine for the hydrogen-bonded tyrosine) similarly leads to specific addition of water and predominant production of hydroxylated products (Criswell et al., 2012; Mafu et al., 2015). In both cases, this contrasts with the occasional addition of water and relatively small amounts of hydroxylated product observed here (Figure 4). Accordingly, it seems unlikely that introducing a single hydroxyl group would be sufficient to both bind and activate a water to largely function as a general base, particularly without adding to the carbocation more often than is observed. Thus, although such a direct mechanism cannot be entirely ruled out, it seems most likely that the observed effects are due to the electrostatic effect of the introduced hydroxyl on stabilization of the ent-pimar-15-en-8-yl⁺ for deprotonation. The water bound in the cavity created by substitution of Ala for this Ile would then similarly enable the effects on product outcome observed here with such I→A mutants of KSs (Figures 3 and 4). In this mechanism the general base is most likely the pyrophosphate anion co-product, as previously suggested for class I terpene synthases in general (Pemberton and Christianson, 2016).

Regardless of the exact role of this key Ile, the conservation of this residue across all known embryophyta KSs is consistent with the hypothesis that the earliest plant terpene synthase was a bifunctional CPS/KS (e.g., much like the PpCPS/KS studied here). This hypothesis is further reinforced by the observation that the key Ile from plant KSs is not found in the functionally analogous enzymes that have been identified in microbes (Morrone et al., 2009). In particular, this Ile is not conserved in the bifunctional CPS/KS found in certain fungi (Bomke and Tudzynski, 2009), which otherwise nominally resemble those found in plants (Peters, 2013). Nor is this Ile conserved in the known single-domain monofunctional KSs in bacteria, where such activity appears to have arisen via parallel evolution in two separate contexts (Morrone et al., 2009; Smanski et al., 2011; Hershey et al., 2014; Lu et al., 2015). The widespread functional conservation of a key Ile shown here indicates an early appearance of KSs in plant evolution, which is consistent with the importance of ent-kaurene derived molecules in all embryophyta, even the bryophytes that do not produce gibberellins (Hirano et al., 2007; Yasumura et al., 2007). Such early origins for KSs also is consistent with the previously suggested staggered evolution of gibberellin biosynthesis in which modification of the ent-kaurane backbone into gibberellins only arose after divergence of the bryophytes from tracheophytes (Zi et al., 2014). Moreover, the catalytic plasticity of these enzymes demonstrated here is further consistent with the hypothesis that the KSs gave rise to the plant family of classic (class I) terpene synthases more generally, the importance of which in the diversification of plant metabolism is highlighted by their presence as moderately large gene families in all tracheophytes (Chen et al., 2011).

MJ designed and conducted the experiments. MJ and RP jointly conceived of the project, interpreted the data, and wrote the paper.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.