L-Malate (-2) protonation state is required for efficient decarboxylation to L-lactate by the malolactic enzyme of Oenococcus oeni

Abstract

Malolactic fermentation (MLF) is responsible for the decarboxylation of L-malic into lactic acid on most red wines and some white wines. It reduces the acidity of wine, improves flavor complexity and microbiological stability. Despite its industrial interest, the MLF mechanism is not fully understood. The objective of this study was to provide new insights into the role of pH on the binding of malic acid to the malolactic enzyme (MLE) of Oenococcus oeni. The latter has an optimum activity on the 3.0-6.0 pH range. To this task, sequence similarity networks and phylogenetic analysis were used to generate an MLE homology model, which was further refined by molecular dynamics simulations. The resulting model, together with quantum polarized ligand docking (QPLD), was used to describe the MLE binding pocket and pose of L-malic acid (MAL) and its L-malate (-1) and (-2) protonation states (MAL-1 and MAL-2, respectively). MAL-2 has the lowest ∆Gbinding, followed by MAL-1 and MAL, with values of -5.7, -4.7 and -3.5 kcal/mol, respectively, consistent with those obtained by ITC assays. Furthermore, molecular dynamics and MM/GBSA results suggest that only MAL-2 displays an extended open conformation at the binding pocket, satisfying the geometrical requirements for Mn+2 coordination, a critical component of MLE activity. Altogether, these results contribute to the understanding of the role of pH on the Oenococcus oeni malolactic enzyme activity and the malolactic fermentation, as a whole.