BBMB Research Seminars

March 22

Sladjana Prisic
Department of Biochemistry, Biophysics, and Molecular Biology
Iowa State University
Copalyl diphosphate synthase (CPS) - a model class II diterpene cyclase
1414 Molecular Biology Buidling
4:10 p.m.


Terpene synthases or cyclases are intriguing group of enzymes that conduct cyclization of simple linear substrates utilizing two basic mechanisms – ionization initiated (class I) and protonation initiated (class II) reaction. The core carbon backbone is further decorated by various functional groups giving around 50,000 known terpenoid structures and creating a significant pool of physiologically relevant and pharmaceutically interesting compounds.

Biosynthesis of labdane-related diterpenes (which contains more than 5,000 members) from the common precursor geranylgeranyl pyrophosphate (GGPP) is initiated by a poorly understood protonation of an olefin and subsequent cyclization, known as class II mechanism. We chose copalyl diphosphate synthase (CPS), the enzyme that catalyses committed step in gibberellin and phytoalexin biosynthesis, as a model system for class II diterpene cyclization. We cloned and functionally expressed CPS enzymes from Arabidopsis, rice, and corn. Various biochemical and molecular biology techniques were used to determine their function and biochemical characteristics, including the expression patterns and product specificity, as well as stabilization for in vitro studies. Additionally, we conducted more detailed biochemical analysis of Arabidopsis CPS (AtCPS) to optimize the enzymatic assay and discovered a novel intra-steric regulation by Mg2+ in synergism with GGPP that may be involved in control of gibberellin biosynthesis. Getting further into the underlying mechanism, we used mutagenesis, inhibitors, and substrate analogues that helped us identify residues essential for catalysis and metal cofactor binding. Specifically, an epoxy analog of GGPP was coupled to site-directed mutagenesis to provide evidence that the DXDD motif, with the assistance of conserved His331, in class II diterpene cyclases is involved in C=C double bond protonation, similar to the role of the DXDD motif found in the otherwise unrelated triterpene cyclases. We also showed that the conserved DXDD motif in AtCPS is a potential inhibitory binding site for Mg2+ and GGPP. Finally, we conducted domain-swapping experiments of class I and class II diterpene cyclases (i.e. kaurene synthase and copalyl synthase), as well as CPSs from rice with different product stereochemistry (OsCPS2ent and OsCPS4syn), in order to identify structural requirements for class II catalysis.

In conclusion, our findings created a strong basis for further mechanistic and structural studies of the significant class II terpene synthases that is the first and the most important step towards understanding of terpene, especially labdane biosynthesis. In addition, by resolving class II terpene cyclase mechanism, we are creating a powerful tool to engineer synthesis of many pharmaceutically interesting compounds in the future.