Research Article: Computational study on a puzzle in the biosynthetic pathway of anthocyanin: Why is an enzymatic oxidation/ reduction process required for a simple tautomerization?

Date Published: June 13, 2018

Publisher: Public Library of Science

Author(s): Hajime Sato, Chao Wang, Mami Yamazaki, Kazuki Saito, Masanobu Uchiyama, Sara Amancio.


In the late stage of anthocyanin biosynthesis, dihydroflavonol reductase (DFR) and anthocyanidin synthase (ANS) mediate a formal tautomerization. However, such oxidation/reduction process requires high energy and appears to be unnecessary, as the oxidation state does not change during the transformation. Thus, a non-enzymatic pathway of tautomerization has also been proposed. To resolve the long-standing issue of whether this non-enzymatic pathway is the main contributor for the biosynthesis, we carried out density functional theory (DFT) calculations to examine this non-enzymatic pathway from dihydroflavonol to anthocyanidin. We show here that the activation barriers for the proposed non-enzymatic tautomerization are too high to enable the reaction to proceed under normal aqueous conditions in plants. The calculations also explain the experimentally observed requirement for acidic conditions during the final step of conversion of 2-flaven-3,4-diol to anthocyanidin; a thermodynamically and kinetically favorable concerted pathway can operate under these conditions.

Partial Text

Anthocyanins, an important plant secondary metabolites, contribute to the diversity of colored pigments in plants, especially in flowers and fruits, and also act as photo-protectants, [1] visual signals [2] for insects to promote pollination, and antioxidants. [3–6] Although anthocyanin biosynthesis has been extensively studied, [7, 8] some details still remain unclear. In the late stage of anthocyanin biosynthesis, oxidizing and reducing enzymes appear to be necessary for the conversion of dihydroflavonol to anthocyanidin [9, 10] (Fig 1 Route A).

All calculations were performed with Gaussian 09 [18] and GRRM11 [19–23] programs. Structure optimization and frequency calculation were done with the M06-2X/6-31G(d,p) method. [24, 25] Solvation was evaluated by the self-consistent reaction field (SCRF) method using the polarizable continuum model (PCM). Single point energy was calculated at the MP2/6-311++G(d,p) level based on the M06-2X-optimized structure, since M06-2X is known to be inappropriate for describing the relative energies of proton transfer reactions. [26] Relative Gibbs free energy energies (ΔGrel) based on single point energy at the MP2 level and frequency calculation at the M06-2X level are given for all discussions.

The optimized structures (without any symmetry assumptions) and energies of all CPs (complexes), TSs (Transition States), and the product in the conversion of dihydroflavonol to 2-flaven-3,4-diol are shown in Fig 2.




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