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

Abstract

The naphthyridinomycin biosynthesis enzyme NapI selectively performs the desaturation of a free L-arginine amino acid at the C 4 −C 5 bond as part of its antibiotic biosynthesis reaction. This is an unusual reaction triggered by a nonheme iron dioxygenase as most L-Arg activating nonheme iron enzymes cause substrate hydroxylation at an aliphatic C−H bond; hence, this reaction has great potential in biotechnology for the efficient synthesis of drug and fragrance molecules. However, desaturation reactions in chemical catalysis are challenging reactions to perform as they often require toxic heavy metals and solvents. Its enzymatic biosynthesis would provide an environmentally benign alternative. To find the biotechnological application of NapI, we performed a computational study on the enzyme. However, the catalytic mechanism of L-Arg desaturation by NapI is controversial and several possible mechanisms have been suggested via either radical or charge-transfer pathways. We set up an enzymatic structure from the deposited crystal structure coordinates of substrate-bound NapI and inserted co-substrate (α-ketoglutarate) and solvated the structure in a water environment. Thereafter, we set up a series of quantum mechanics/molecular mechanics calculations and validated the results against experimental data. Subsequently, we investigated the mechanisms leading to C 5 -and C 4 -hydroxylation and C 4 −C 5 -desaturation of L-Arg via radical and charge-transfer pathways. The calculations give a rate-determining hydrogen atom abstraction step that is lowest for the C 5 −H position and gives a radical intermediate, although the hydrogen atom abstraction from the C 4 −H group is less than ΔG = 2 kcal mol −1 higher in energy. The calculations show that isotopic substitution of key C−H bonds with C−D changes the product distributions dramatically. The C 5 radical intermediate gives bifurcation pathways with a small second hydrogen atom abstraction from the C 4 −H group and a much higher OH rebound barrier. We also located a charge-transfer intermediate of an iron(II)-hydroxo species with a cationic substrate, but its kinetics and thermodynamics with respect to the radical intermediate make it an unviable mechanism. A comparison with alternative hydroxylating enzymes identifies key differences in substrate orientation and positioning and their second coordination sphere interactions with protein that induces a different dipole and electric field direction. Our work shows that the desaturation of L-Arg is governed by the substrate-binding orientation and the polarity and hydrogen bonding interactions in the substrate-binding pocket that guides the reaction to desaturation products by locking the iron(III)-hydroxo group in position. Our understanding has given valuable insight into enzymatic reactivity and may help to design and engineer enzymes better for highly selective reaction processes in biotechnology.

An Active Site Tyr Residue Guides the Regioselectivity of Lysine Hydroxylation by Nonheme Iron Lysine-4-hydroxylase Enzymes through Proton-Coupled Electron Transfer

“…By contrast, in the naphthyridinomycin biosynthesis enzyme NapI, the dipole is along the substrate backbone and appear not to influence the C−H bond strengths in the substrate. 117 As a consequence, in NapI, the weakest C−H bond in the gas phase is activated by the protein, whereas in VioC, one of the stronger bonds is activated instead. The dipole moment in model C is also perpendicular to the aromatic ring of Tyr 233 and hence is likely to stabilize a radical center on this group and trigger an electron transfer from Tyr 233 to iron(IV)-oxo and a simultaneous proton transfer from Tyr 233 to Asp 179 .…”

“…In VioC, the dipole moment therefore guides the reaction selectivity and triggers a lesser thermodynamic favorable pathway. By contrast, in the naphthyridinomycin biosynthesis enzyme NapI, the dipole is along the substrate backbone and appear not to influence the C–H bond strengths in the substrate . As a consequence, in NapI, the weakest C–H bond in the gas phase is activated by the protein, whereas in VioC, one of the stronger bonds is activated instead.…”

CO2 Reduction by an Iron(I) Porphyrinate System: Effect of Hydrogen Bonding on the Second Coordination Sphere

“…In particular, single-point calculations at the UB3LYP-GD3/BS2 level of theory were run in Gaussian with an electric field located along the molecular x , y , or z axis, with magnitudes ranging from −200 to +200 au, and the results are shown in Figure . These electric-field perturbations were used previously in our group and shown to influence charge distributions in complexes and bifurcation patterns in chemical catalysis. , In particular, recent work showed that charged groups in proteins can influence the strength of the C–H bonds in substrates and direct a reaction selectivity to a specific bond in a substrate. , Thus, an electric-field perturbation has a major effect on the thermodynamics for proton transfer and the reaction energy. To be specific, an electric-field effect along the negative z axis is along the proton-transfer axis and makes the first proton-transfer step more exergonic, while a field in the opposite direction makes the reaction more endergonic, i.e., less likely.…”

Substrate-Dependent Mechanism Switch in the Desaturation Reactions of the Mononuclear Nonheme Iron Enzyme PtlD

“…For instance, studies on NvfI, TqaL, AspE, KabC, PIsnB, and PvcB have reported such findings. Since the feasibility of single ET between the substrate radical and Fe III –OH [in pathway (c)] is debatable from the thermodynamic perspective, − we should still be cautious regarding the detailed steps in certain cases. The uncertainty may be attributed to factors such as the redox potential of Fe III –OH, the ET barrier between the substrate radical and cation intermediate, the incompatibility of an unstable cation intermediate in the enzyme binding pocket, and the structural properties of substrates.…”