Fine-tuning of microsolvation and hydrogen bond interaction regulates substrate channelling in the course of flavonoid biosynthesis

In the course of metabolite formation, some multienzymatic edifices, the so-called metabolon, are formed and lead to a more efficient production of these natural compounds. One of the major features of these enzyme complexes is the facilitation of direct transfer of the metabolite between enzyme active sites by substrate channelling. Biophysical insights into substrate channelling remain scarce because the transient nature of these macromolecular complexes prevents the observation of high resolution structures. Here, using molecular modelling, we describe the substrate channelling of a flavonoid compound between DFR (dihydroflavonol-4-reductase) and LAR (leucoanthocyanidin reductase). The simulation presents crucial details concerning the kinetic, thermodynamic, and structural aspects of this diffusion. The formation of the DFR–LAR complex leads to the opening of the DFR active site giving rise to a facilitated diffusion, in about 1 μs, of the DFR product towards LAR cavity. The theoretically observed substrate channelling is supported experimentally by the fact that this metabolite, i.e. the product of the DFR enzyme, is not stable in the media. Moreover, along this path, the influence of the solvent is crucial. The metabolite remains close to the surface of the complex avoiding full solvation. In addition, when the dynamic behaviour of the system leads to a loss of interaction between the metabolite and the enzymes, water molecules through bridging H-bonds prevent the former from escaping to the bulk.