COMPARATIVE STRUCTURAL AND BIOPHYSICAL ANALYSIS OF CHALCONE SYNTHASE (CHS) AND CHALCONE ISOMERASE-LIKE (CHIL) PROTEINS ACROSS PLANT LINEAGES

Author

• Arman Ahmed, Kennesaw State UniversityFollow

Semester of Graduation

Spring 2026

Degree Type

Thesis

Degree Name

MASTER OF SCIENCE IN CHEMICAL SCIENCES

Department

DEPARTMENT OF CHEMISTRY & BIOCHEMISTRY

Committee Chair/First Advisor

Dr. SOON GOO LEE

Second Advisor

Dr. MICHAEL VAN DYKE

Third Advisor

Dr. ANIMESH V. ADITYA

Abstract

Chalcone synthase (CHS) and chalcone isomerase-like (CHIL) proteins are central regulators of the flavonoid biosynthetic pathway in land plants. CHS catalyzes the committed step in flavonoid biosynthesis, while CHIL, a catalytically inactive paralog of chalcone isomerase, reduces CHS promiscuity and enhances pathway flux toward naringenin chalcone. Despite growing evidence for CHIL's regulatory importance, comparative structural and biophysical data across evolutionarily diverse plant lineages remain limited. This thesis presents a structural and biophysical investigation of CHS and CHIL proteins from six plant species spanning the major divisions of land plant evolution: Glycine max, Oryza sativa, Gossypium hirsutum, Solanum lycopersicum, Selaginella moellendorffii, and Marchantia polymorpha. Apo crystal structures of OsCHIL and SmoCHIL were determined, representing the first structural characterizations of CHIL from a monocot and a lycophyte. A homology model of GmCHIL and a GmCHIL-GmCHS complex model were constructed and used for molecular docking of seven flavonoid ligands. Thermal shift assays confirmed folding quality across all six CHIL orthologs and identified naringenin chalcone as the most promising GmCHIL ligand candidate. Binding assays validated a direct GmCHIL-GmCHS interaction with an apparent K_d of ~5.8-6.6 µM. Phylogenetic analysis confirmed conservation of key functional residues across approximately 400 million years of plant evolution. Together, these results establish a structural and biophysical framework for understanding how CHIL proteins from evolutionarily distinct plant lineages regulate flavonoid biosynthesis.