Characterization of three cytochrome P450 reductases from sorghum bicolor

Authors: ZHANG, BIXIA - Washington State University; RALPH, JOHN - University Of Wisconsin; DAVYDOV, DMITRI - Washington State University; VERMERRIS, WILFRED - University Of Florida; Sattler, Scott; KANG, CHULHEE - Washington State University

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/17/2022
Publication Date: 4/1/2022
Citation: Zhang, B., Ralph, J., Davydov, D.R., Vermerris, W., Sattler, S.E., Kang, C. 2022. Characterization of three cytochrome P450 reductases from sorghum bicolor. Journal of Biological Chemistry. 298(4):1-20. https://doi.org/10.1016/j.jbc.2022.101761.
DOI: https://doi.org/10.1016/j.jbc.2022.101761

Interpretive Summary: In the U.S., sorghum is a major drought tolerant grain and forage crop, which produces numerous compounds that require a class of enzymes called NADPH-dependent cytochrome P450 reductases (CPRs). In sorghum, CPRs are necessary for plant cell wall synthesis and production of red pigments among many other pathways. To understand the structure and function of three CPRs from sorghum, SbCPR2a, SbCPR2b and SbCPR2c, these proteins were subjected to X-ray crystallography, and enzyme activity was measured under various conditions. The enzyme activity showed that all three sorghum CPRs function in the synthesis of the cell wall polymer lignin. The SbCPR2b structure was compared to CPRs of mammals, which enabled the researchers from across the U.S. to identify critical portions of the protein, which suggested that its binding domain may be more flexible than the mammalian ones. Overall, the hinge region of the SbCPR2 protein could serve as a target to alter biomass composition for bioenergy and forage uses through protein engineering.

Technical Abstract: Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multi-domain enzyme that donates electrons for the hydroxylation steps catalyzed by the Class II cytochrome P450 enzymes involved in the synthesis of many primary and secondary metabolites, which include the enzymes trans-cinnamate-4-hydroxylase (C4H), p-coumarate-3'-hydroxylase (C3'H) and ferulate-5-hydroxylase (F5H) involved in monolignol biosynthesis. Because of its critical role in monolignol biosynthesis, alterations of CPR activity could change in overall composition and amount of lignin. To understand the structure and function of three CPRs from sorghum, SbCPR2a, SbCPR2b and SbCPR2c, the recombinant proteins were subjected to X-ray crystallography and steady-state kinetics under various conditions were measured. The steady-state kinetic analyses demonstrated that they all supported the oxidation step of SbC4H1 and SbC3'H. Comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance, and suggest that the plant flavin mononucleotide-binding domain might be more flexible than mammalian ones. The structure of SbCPR2b included the first observation of NADP+ in native CPR. Overall, the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage uses through protein engineering.