Computational Survey of Recent Experimental Developments in the Hydroxylation Mechanism of Kynurenine 3-Monooxygenase

Recently, two new mechanistic proposals for the kynurenine 3-monooxygenase (KMO) catalyzed hydroxylation reaction of L-Kynurenine (L-Kyn) have been proposed. According to the first proposal, instead of the distal oxygen, the proximal oxygen of the hydroperoxide intermediate of flavin adenine dinucleotide (FAD) is transferred to the substrate ring. The second study proposes that L-Kyn participates in its base form in the reaction. To address these proposals, the reaction was reconsidered with a 386 atom quantum cluster model that is based on a recent X-ray structure (PDB id: 6FOX). The computations were carried out at the UB3LYP/6-311+G- (2d,2p)//UB3LYP/6-31G(d,p) level with solvation (polarizable continuum model) and dispersion (DFT-D3(BJ)) orrections. To supplement the results of the density functional theory (DFT) calculations, molecular dynamics (MD) simulations of the protein−substrate complex were employed. The comparison of a proximal oxygen transfer mechanism to the distal oxygen transfer mechanism revealed that the former requires too high of a barrier energy while the latter validated our previous results. According to the MD simulations, the hydroperoxy moiety does not favor an alignment that might promote the proximal oxygen transfer mechanism. In the second part of the study, hydroxylation reaction with the base form of L-Kyn was sought. Although DFT calculations confirmed a much more facile reaction with the base form of L-Kyn, a mechanism which would allow the deprotonation of the L-Kyn before the oxygen transfer could not be determined with the X-ray-based positions. A concerted mechanism with both the oxygen transfer and the deprotonation required a high barrier energy. A stepwise mechanism involving the deprotonation of LKyn was found, starting from an MD frame. The overall barrier of the oxygen transfer step of this model was found to be in the range of that of with neutral L-Kyn. MD simulations supported the idea of ineffectiveness of the nearby shell surrounding the utilized active site core on the deprotonation of L-Kyn..