Structural Similarities and Overlapping Activities among Dihydroflavonol 4-Reductase, Flavanone 4-Reductase, and Anthocyanidin Reductase Offer Metabolic Flexibility in the Flavonoid Pathway

Authors: LEWIS, JACOB - Washington State University; ZHANG, BIXIA - Washington State University; HARZA, RISHI - Washington State University; Palmer, Nathan - Nate; Sarath, Gautam; Sattler, Scott; TWIGG, PAUL - University Of Nebraska; VERMERRIS, WILFRED - University Of Florida; KANG, CHULHEE - Washington State University

Submitted to: International Journal of Molecular Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 9/5/2023
Publication Date: 9/9/2023
Citation: Lewis, J.A., Zhang, B., Harza, R., Palmer, N.A., Sarath, G., Sattler, S.E., Twigg, P., Vermerris, W., Kang, C. 2023. Structural Similarities and Overlapping Activities among Dihydroflavonol 4-Reductase, Flavanone 4-Reductase, and Anthocyanidin Reductase Offer Metabolic Flexibility in the Flavonoid Pathway. International Journal of Molecular Sciences. Volume 24, Issue 18, p. 13901. https://doi.org/10.3390/ijms241813901.
DOI: https://doi.org/10.3390/ijms241813901

Interpretive Summary: Plants synthesize many flavonoid compounds, which are often red or purple in color and serve wide range of functions including their strong antioxidant properties. Enzymes involved in making these compounds are good targets for engineering the production of new flavonoid compounds to improve plant and human health. In this report, three enzymes, dihydroflavonol 4-reductase, flavonone 4-reductase, and anthocyanidin reductase from sorghum and swichgrass were analyzed and their protein structures solved to understand how these enzymes function in synthesis of flavonoid compounds. Detailed analysis determined the critical groups required for enzyme activity. This study points to ways in which the flavonoid pathway can be modified to improve the production of the flavonoids beneficial to animal and human health.

Technical Abstract: 3-Deoxyanthocyanidins (3-DOA) are potent antioxidants, which play a key role in defense against pathogens through the formation small oily droplets at the site of invasion that can inhibit pathogen growth. 3-DOAs are derived from flavon-(3)-4-(di)ols. Dihydroflavonol 4-reductase (DFR) and flavanone 4-reductase (FNR) catalyze the reduction of dihydroflavonols and flavanones using NAD(P)H to produce various flavon-(3)-4-(di)ols in the central steps of flavonoid synthesis. In addition to their shared amino acid sequence similarity, the 3D structures of DFR and FNR from sorghum and switchgrass showed structural similarities, including a continuous NADPH-binding Rossman-fold domain and substrate-binding motif. The catalytic mechanism and substrate specificity of the enzymes were deduced from crystal structures, site-directed mutagenesis and kinetic and thermodynamic analyses. In both switchgrass DFRa (PvDFRa) and sorghum FNR (SbFNR), the nicotinamide moiety was properly located for the pro-R hydride transfer towards the substrate, together with acid-base catalysis of the nearby catalytic triad, and both NADPH and NADH serve as a coenzyme. Although DFR displayed higher activity for dihydroflavonols, it also showed significant activity for flavanones and was inhibited by the flavonol quercetin. FNR did not show any activity against dihydroflavonols, but displayed marginal activity against flavanones. Both DFR and FNR have a high degree of structural similarity with anthocyanidin reductase (ANR), and both SbFNR1 and SbFNR2 displayed significant ANR activity reducing 3-DOA to flavan-3-ols. Thus, SbFNR1 and SbFNR2 might be ANR instead of FNR. Through enzymatic analyses and comparisons among the crystal structures of grape DFR / ANR, switchgrass DFR and sorghum DFR / FNR / ANR, a key signature sequences and basis for future metabolic engineering of flavonoid metabolism are presented through insights such as the increased catalytic efficiency of DFR enzymes in which specific amino acid residues were replaced.