Catalytic activity and interaction among chalcone synthase, chalcone isomerase, and chalcone isomerase-like-protein

Authors: LEWIS, JACOB - Washington State University; JACOBO, ERIC - Washington State University; Palmer, Nathan - Nate; VERMERRIS, WILFRED - University Of Florida; Sattler, Scott; BROZIK, JAMES - Washington State University; Sarath, Gautam; KANG, CHULHEE - Washington State University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2024
Publication Date: 5/22/2024
Citation: Lewis, J.A., Jacobo, E.P., Palmer, N.A., Vermerris, W., Sattler, S.E., Brozik, J.A., Sarath, G., Kang, C. 2024. Catalytic activity and interaction among chalcone synthase, chalcone isomerase, and chalcone isomerase-like-protein. International Journal of Molecular Sciences. 25(11). Article 5651.. https://doi.org/10.3390/ijms25115651.
DOI: https://doi.org/10.3390/ijms25115651

Interpretive Summary: Flavonoids are reddish, purple pigments present in many plants that have potential applications in medicine. Flavonoids may possess a number of medical benefits, including anticancer, antioxidant, anti-inflammatory, and antiviral properties. In this study, Chalcone synthase (CHS) and chalcone isomerase (CHI) enzymes, which are responsible for the first two steps of flavonoid synthesis were examined from grasses, an untapped source of these pigments. Examination of these enzyme structures and how these two enzymes interact with each other showed their interaction greatly increased the efficiency of flavonoids synthesis. These findings indicate the importance of these interactions in determining the amounts and types of flavonoids pigments produced in plant cells, which play important roles for protecting plants from UV light, pests and diseases. The U.S. flavonoid market was value at $1.23 billion in 2021, and this research is an initial step to producing flavonoids in crop such as corn and sorghum to benefit human health.

Technical Abstract: Chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the first two committed steps of the flavonoid pathway that plays a pivotal role in the growth and reproduction of land plants, including UV protection, pigmentation, symbiotic nitrogen fixation, and pathogen resistance. Based on the obtained X-ray crystal structures of CHS, CHI, and chalcone isomerase-like protein (CHIL) from the same monocotyledon, Panicum virgatum, along with the results of the steady-state kinetics, spectroscopic/thermodynamic analyses, intermolecular interactions, and their effect on each catalytic step are proposed. In addition, PvCHI’s unique activity for both naringenin chalcone and isoliquiritigenin was analyzed, and the observed hierarchical activity for those type-I and -II substrates was explained with the intrinsic characteristics of the enzyme and two substrates. The structure of PvCHS complexed with naringenin supports uncompetitive inhibition. PvCHS displays intrinsic catalytic promiscuity, evident from the formation of p-coumaroyltriacetic acid lactone (CTAL) in addition to naringenin chalcone. In the presence of PvCHIL, conversion of p-coumaroyl-CoA to naringenin through PvCHS and PvCHI displayed ~400-fold increased Vmax with reduced formation of CTAL by 70%. Supporting this model, molecular docking, ITC (Isothermal Titration Calorimetry), and FRET (Fluorescence Resonance Energy Transfer) indicated that both PvCHI and PvCHIL interact with PvCHS in a non-competitive manner, indicating the plausible allosteric effect of naringenin on CHS. Significantly, the presence of naringenin increased the affinity between PvCHS and PvCHIL, whereas naringenin chalcone decreased the affinity, indicating a plausible feedback mechanism to minimize spontaneous incorrect stereoisomers. These are the first findings from a three-body system from the same species, indicating the importance of the macromolecular assembly of CHS-CHI-CHIL in determining the amount and type of flavonoids produced in plant cells.