A QM/MM Free Energy Study of the Oxidation Mechanism of Dihydroorotate Dehydrogenase (Class 1A) from <i>Lactococcus lactis</i>

The dihydroorotate dehydrogenase (DHOD) class 1A enzyme catalyzes is the only redox enzyme in the biosynthetic pathway (de novo) of pyrimidines where dihydroorotate (DHO) is oxidized to orotate (OA) coupled to reduction of a flavin mononucleotide (FMN) cofactor. The rupture of two DHO C–H bonds can proceed in a concerted or stepwise way. Herein, the catalytic mechanism of DHOD from <i>Lactococcus lactis</i> involving DHO oxidation (first half-reaction) was described using a hybrid quantum mechanics/molecular mechanics (QM/MM) approach and molecular dynamics simulations. The free energy profile obtained from self-consistent charge–density functional tight binding/molecular mechanics calculations (corrected by DFT/MM) reveals that this occurs with the proton abstraction from DHO C₅ to Cys₁₃₀ deprotonated and DHO H₆ is transferred to FMN N₅ in a concerted mechanism with a very low barrier of 5.64 kcal/mol. Finally, through a residual decomposition analysis, the residues that have the main influence on the redox reaction were identified