How Does the Nonheme Iron Enzyme NapI React through l-Arginine Desaturation Rather Than Hydroxylation? A Quantum Mechanics/Molecular Mechanics Study

Thenaphthyridinomycin biosynthesis enzyme NapI selectively performsthe desaturation of a free l-arginine amino acid at the C-4-C-5 bond as part of its antibiotic biosynthesisreaction. This is an unusual reaction triggered by a nonheme irondioxygenase as most l-Arg activating nonheme iron enzymescause substrate hydroxylation at an aliphatic C-H bond; hence,this reaction has great potential in biotechnology for the efficientsynthesis of drug and fragrance molecules. However, desaturation reactionsin chemical catalysis are challenging reactions to perform as theyoften require toxic heavy metals and solvents. Its enzymatic biosynthesiswould provide an environmentally benign alternative. To find the biotechnologicalapplication of NapI, we performed a computational study on the enzyme.However, the catalytic mechanism of l-Arg desaturation byNapI is controversial and several possible mechanisms have been suggestedvia either radical or charge-transfer pathways. We set up an enzymaticstructure from the deposited crystal structure coordinates of substrate-boundNapI and inserted co-substrate (& alpha;-ketoglutarate) and solvatedthe structure in a water environment. Thereafter, we set up a seriesof quantum mechanics/molecular mechanics calculations and validatedthe results against experimental data. Subsequently, we investigatedthe mechanisms leading to C-5- and C-4-hydroxylationand C-4-C-5-desaturation of l-Argvia radical and charge-transfer pathways. The calculations give arate-determining hydrogen atom abstraction step that is lowest forthe C-5-H position and gives a radical intermediate,although the hydrogen atom abstraction from the C-4-Hgroup is less than & UDelta;G = 2 kcal mol(-1) higher in energy. The calculations show that isotopic substitutionof key C-H bonds with C-D changes the product distributionsdramatically. The C-5 radical intermediate gives bifurcationpathways with a small second hydrogen atom abstraction from the C-4-H group and a much higher OH rebound barrier. We alsolocated a charge-transfer intermediate of an iron(II)-hydroxo specieswith a cationic substrate, but its kinetics and thermodynamics withrespect to the radical intermediate make it an unviable mechanism.A comparison with alternative hydroxylating enzymes identifies keydifferences in substrate orientation and positioning and their secondcoordination sphere interactions with protein that induces a differentdipole and electric field direction. Our work shows that the desaturationof l-Arg is governed by the substrate-binding orientationand the polarity and hydrogen bonding interactions in the substrate-bindingpocket that guides the reaction to desaturation products by lockingthe iron(III)-hydroxo group in position. Our understanding has givenvaluable insight into enzymatic reactivity and may help to designand engineer enzymes better for highly selective reaction processesin biotechnology.