Mechanistic basis for the enantioselectivity of the anaerobic hydroxylation of alkylaromatic compounds by ethylbenzene dehydrogenase

Ethylbenzene Dehydrogenase and Related Molybdenum Enzymes Involved in Oxygen-Independent Alkyl Chain Hydroxylation

“…This suggests that while the removal of a proton from the methylene group is rate limiting for the reaction, the mechanism is strongly influenced by the protonation state of active-site amino acids [Szaleniec et al, 2010]. The origin of the oxygen atom in 1-phenylethanol was confirmed by catalytic experiments with 18 O-labeled water by mass spectrometry analysis of the product, indicating that the labeled oxygen atom is transferred to the product alcohol [Ball et al, 1996;Szaleniec et al, 2014]. Initial kinetic tests with H 2 18 O have not revealed any significant kinetic isotope effects.…”

“…Further calculations using an expanded QM part to avoid over-polarization effects and embedding via EE confirmed the probable involvement of a carbocation intermediate (rather than a radical), as suggested by the initial studies based on kinetic and QSAR analysis. Moreover, the strong overall electrostatic stabilization introduced by incorporating the protein milieu decreased the value of the TS2 barrier, which no longer competes with TS1 for controlling the reaction rate [Borowski et al, 2015;Szaleniec et al, 2014]. Therefore, after the substrate is activated to the level of TS1, there should be no further significant barriers, predicting a very short half-life of the intermediate state.…”

QM and QM/MM Methods Compared

“…Using QM/MM techniques, there is no need to fix any atoms in the periphery of the QM model and in theory there is a more realistic system created. This approach also allowed computational chemists to address the very important question of reaction enantioselectivity, both introduced by appropriate substrate binding (like in the case of the short-chain alcohol dehydrogenases where appropriate binding of the alcohol/ketone with respect to NAD + /NADH and Tyr H-bond donor in the during reactions, leading ES complex is responsible for reaction enantioselectivity ) or due to differences in the steric interactions during reactions, leading to different heights of the energy barriers observed for enantiomers or prochiral reagents (e.g., ethylbenzene dehydrogenase (EbDH) (Szaleniec et al, 2014) or cyclohexanone monooxygenase (Polyak, Reetz, & Thiel, 2012). …”

“…The molecular mechanism of these hydroxylations is very interesting as the whole process proceeds without molecular oxygen due to the anaerobic nature of the bacteria metabolism. These enzymes are also very interesting from the point of view of potential application as they exhibit high regioselectivity and EbDH is also highly enantioselective (+95% enantiomeric excess for more than 30 substrates) (Dudzik et al, 2013;Szaleniec et al, 2014). It was shown by isotope-labeling studies that oxygen is derived from a water molecule while the catalytic cofactor is oxidized by an external electron acceptor via enzyme's metal clusters including a high potential heme cofactor.…”

“…The approximate energy difference between radical and carbocation solution was estimated to be 70 kJ/mol (calculated as the difference between Mo(V) triplet and Mo(IV) singlet solution). As a result, the QM2:MM/EE calculations seem to provide the best available (up to date) description of the EbDH reactivity (Szaleniec et al, 2014).…”