Proposal Defense – Min Wang
About the event
Speaker: Min Wang
Group: ChulHee Kang
Title: The Structural and Functional Characterization of P450 Enzymes in the Monolignol and Flavonoid Pathways of Sorghum bicolor
Abstract:
Sorghum bicolor is a drought- and heat-tolerant cereal crop and an attractive lignocellulosic feedstock whose value depends critically on how carbon is partitioned through the phenylpropanoid pathway into lignin and flavonoid metabolites. ER-membrane-bound cytochrome P450 monooxygenases—including the cinnamate 4-hydroxylase isoenzymes (C4H1–3), p-coumarate 3′-hydroxylase (C3′H), ferulate 5-hydroxylase (F5H), flavonoid 3′-hydroxylase (F3′H), flavonoid 3′,5′-hydroxylase (F3′5′H), and flavone synthase II (FNSII)—work in concert with multiple NADPH–cytochrome P450 reductase isoforms (SbCPR2a, 2b, 2c) to control lignin composition, pigmentation, and stress adaptation. However, with the exception of C4H1, the structural basis of substrate recognition, isoform-specific catalysis, and CPR coupling in sorghum P450s remains largely unknown. This proposal aims to develop a structure–function framework for these six P450s and their CPR partners in the monolignol and flavonoid pathways. Objective 1 will characterize the structural properties of C4H2 and C4H3 and quantify how each isoenzyme pairs with the three SbCPR isoforms using reconstituted liposomal monooxygenase systems and steady-state kinetics. Objective 2 will cover C3′H and F5H, combining expression and purification, crystallization, and kinetic profiling to reveal how active-site architecture and CPR docking surfaces tune lignin-branch flux. Objective 3 will characterize F3′H, F3′5′H, and FNSII in the flavonoid pathway and their dependence on CPR isoform context. Our integrated approach that couples macromolecular crystallography, molecular modeling, enzyme kinetics and site-directed mutagenesis could yield a blueprint for engineering biosynthesis for two critical secondary metabolites to enhance bioenergy traits and stress resilience of sorghum.