Plant triterpenoid scaffolding: A tale of two cyclases

Triterpenoids constitute one of the largest and most ubiquitous groups of natural products in the living world with more than 20,000 members, including ginsenosides and the vaccine adjuvant QS-21 as some of the best known examples (Thimmappa et al., 2014). In plants, triterpenoids serve a wide range of functions in both core metabolism, such as sterols in regulating cell membranes, and specialized metabolism, such as saponins in mediating herbivore resistance. Despite this extraordinary structural and functional diversity, all triterpenoids derive from the central precursor 2,3-oxidosqualene thanks to a group of enzymes called 2,3-oxidosqualene cyclases (OSCs). Exactly how the regio- and stereo-selectivities vary from one OSC to the next has intrigued and puzzled plant scientists in the past decades (Cárdenas et al., 2019). In this issue of Plant Physiology, Günther et al. (2022) show that in the biosynthesis of α- and β-amyrin in wintercress (Barbarea vulgaris), the difference in product selectivity is largely determined by only a couple of key amino acids in the OSCs. In a triterpenoid scaffold formation reaction, an OSC protonates 2,3-oxidosqualene to yield a carbocation. Due to its highly unstable chemistry, the carbocation undergoes a structural change cascade, including cyclization and re-arrangement, which are guided and finally terminated by the OSC to form final product(s) (Thimmappa et al., 2014; Cárdenas et al., 2019). No plant OSC structures have been solved, presumably due to difficulties associated with their membrane-bound nature. However, crystal structures of lanosterol synthase and squalene-hopane synthase, two OSCs in human and the bacterium Alicyclobacillus acidocaldarius, respectively, feature similar overall structure despite their very different origins and low (25%) sequence identity (Wendt et al., 1997; Thoma et al., 2004). It is thus reasonable to expect that comparative structure–function analysis using these templates can offer informative insights into the working of plant OSCs. Günther et al. (2022) employed this approach to investigate a pair of closely related OSCs with distinct product profiles in B. vulgaris, namely lupeol synthase 5 G-type (LUP5-G) from the insect-resistant chemotype G (“glabrous”) and LUP5 P-type (LUP5-P) from the more susceptible chemotype P (“pubescent”). Previous works suggest that the G chemotype’s effective self-defense against herbivores is associated with its high abundance of β-amyrin-derived saponins and the ability to make β-amyrin by LUP5-G (Kuzina et al., 2009; Khakimov et al., 2015; Liu et al., 2019). Although both of these OSCs can produce multiple triterpenoid products, including lupeol, α-amyrin, and β-amyrin, LUP5-G mainly produces β-amyrin while the dominant product of LUP5-P is α-amyrin (Figure 1). The well-defined product landscapes and the remarkable 98% sequence identity shared between the LUP5 enzymes make these two OSCs and their associated plant chemotypes an ideal system to study the connection between OSC structures, their product profiles, and the eco-physiological impacts beyond. Major products of two multifunctional OSCs, LUP5-G and LUP5-P, underlying different herbivore resistance in two chemotypes of B. vulgaris. A handful of amino acid residues, including a C-terminal leucine (in LUP5-G) or isoleucine (in LUP5-P) (blue), predispose the enzyme’s active site to favor one product over the other due to steric hindrance. Saponins derived from β-amyrin mediate the robust defensive mechanism of chemotype G against insects. Image adapted from Günther et al. (2022, Figure 6) with the “glabrous” leaves of chemotype G and “pubescent” leaves of chemotype P drawn by Katharine Davis (University of Cambridge). Despite accounting for only 2% of the sequence, the difference between LUP5-G and LUP5-P corresponds to 14 amino acids spanning the N- and the C-termini. To tackle the list of all the potential amino acid residues that affect the product profiles of LUP5-G and LUP5-P, Günther et al. (2022) began by generating a set of OSC chimeras. Each chimeric enzyme is based on LUP5-G or LUP5-P with four or five residues of either the N- or C-terminus in one OSC replaced by their respective counterparts of the other. Analyzing the product profiles arising from the chimeric enzymes, the authors then narrowed down the number of residues to be further examined alone or in combination. These chimeric and point mutated OSCs were expressed in the tobacco relative Nicotiana benthamiana. Data indicated that among the different residues between the two LUP5 enzymes, some C-terminal residues are critical for catalytic activities while a few others in the N-terminus help stabilize the enzymes. From the resulting shortlist of key amino acids, Günther et al. (2022) successfully elucidated an N-terminal residue and a C-terminal residue that are largely responsible for the α-amyrin:β-amyrin product ratio and relative activities of the OSCs. Substituting both residues in LUP5-P with the corresponding residues of LUP5-G was sufficient to switch the α-amyrin-dominant product profile to a β-amyrin-dominant one. Subsequent homology modeling of the LUP5 enzymes using the human lanosterol cyclase as template revealed plausible structural roles of the two amino acid residues in question. According to the models, the C-terminal amino acid shapes the active site in a way that strongly drives the carbocation intermediate to adopt either the α- or the β-amyrin conformation, and the position of this C-terminal residue in turn is influenced by the N-terminal one. The product profile-defining features of the LUP5 enzymes, as revealed by Günther et al. (2022), set off two parallel metabolic trajectories for 2,3-oxidosqualene with very different eco-physiological destinies in the two chemotypes of B. vulgaris. This discovery represents an important step in understanding the biosynthesis of amyrin isomers and derived saponins in plants. It also provides another example of how the product space of plant terpene cyclases can be tailored by a handful of interdependent single mutations (Salmon et al., 2016; Cheema et al., 2017). In this case, knowing how the two OSCs’ product landscapes are shaped may inform future efforts to understand how they emerged in the first place. This knowledge may ultimately help us grasp how the wintercress genus Barbarea acquired a chemical defensive feat unlike anything achieved within the Brassicaceae family — producing saponins. Furthermore, with this study, the possibility of understanding and engineering protein structures and functions via comparative analyses of related variants is again highlighted, especially for enzymes with scarce structural data such as OSCs. Conflict of interest statement. None declared.

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