TRICYCLE · Quantitative exploration of catalytic landscapes underlying the functional divergence of triterpene synthases

Quantitative exploration of catalytic landscapes underlying the functional divergence of triterpene synthases.

Plants produce diverse triterpenes, which provide varied benefits to plants including pests and disease resistance. Many triterpenes also have applications in human health. Enzymes of the family Oxidosqualene cyclase’s (OSC’s) are involved in a major step in the pathway in conversion of acyclic 2,3 Oxidosqualene to cyclic products of diverse stereochemistry. Currently more than 100 different triterpene skeletons are known in nature. In plants, within the OSC family, cycloartenol synthases (CAS) are involved in synthesis of tetracyclic sterol precursor cycloartenol in sterol primary metabolism. Similarly, beta-amyrin synthases (BAS), which are functionally diverged from yet closely related to CAS, are involved in synthesis of pentacyclic triterpene secondary metabolites. Mechanistically, the cyclization reaction in this pair of enzymes proceeds through generation of distinct carbocation’s; CAS generates a protosteryl C(20) cation whereas BAS generates a dammarenyl C(20) cation. Initially, enzyme forces its substrate 2,3OS to adopt a synthase specific conformation (CAS forces 2,3OS to adopt a chair-boat-chair but BAS enforces it to adopt all chair conformation) and then protonation of the epoxide triggers a series of ring forming reactions. During polycyclzation, the enzyme stabilizes intermediate carbocation’s from quenching, induces 1,2 hydride and 1,2 methyl shifts and, a final deprotonation before release of the cyclic product. From an evolutionary perspective, these observations raise the question what mutational changes govern catalytic specificity in triterpene cyclases? More specifically, what are/were the key mutational changes responsible for the conversion of CAS to BAS? To approach this question, our study explores the catalytic landscape, the sequence to mechanism relationship, connecting modern-day CA and BA synthases of plants. Catalytic landscaping is a novel concept that uses multidisciplinary approaches to discern the structural changes that interconnect a pair of recently diverging enzymes. As a first step, we have been able to identify candidate residues that govern catalysis of CAS and BAS enzymes through a combination of phylogenetics, homology modelling and sequence-structure associations. Our analysis focused on sequence variation in the active site (5-angstrom distance around the 2,3OS substrate) from experimentally characterized OSC’s from diverse taxa mapped on protein structure models. At these 8 natural mutations, we designed a an in silico mutant library in the BAS background and create a gene library using structure-based combinatorial protein engineering in yeast using a homologous recombination. Finally, expression of the library of size 28=256 in yeast resulted in identification of the novel mutants with strikingly altered product specificities (lib22-1, 19-3, 22-5, 26-30). Some of these accumulate novel products and initial NIST library search did not reveal any significant hits suggesting these could be novel. Future characterization experiments should help us understand their cyclization mechanism and also to understand how these changes have led to the change in product specificity from β-amyrin synthase activity to the observed activities. In addition, another class of mutant’s (lib6-1 and 4-1) known to make a class of triterpene skeletons different from what seen in lib22-1for instance. While the detailed analysis of the library results is still underway; it is interesting to note that few changes in the active site of BAS results in drastic change in product specificity. Some of the future experiments on these mutants will help us understand how these mutations affected the active site for change in product septicity. Though, we have not been able to see change in product specificity from β-amyrin synthase to cycloartenol synthase activity in a BAS background, but we now understand what stage we are in, in the trajectory of converting a β-amyrin synthase to a cycloartenol synthase or cyocolartenol synthase like.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз