How Does the Nonheme Iron Enzyme NapI React Through L-Arginine Desaturation Rather Than Hydroxylation? A QM/MM Study.

Research output: Contribution to journalArticlepeer-review

Abstract

The naphthyridinomycin biosynthesis enzyme NapI selectively performs the desaturation of a free L-arginine amino acid at the C4−C5 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 give 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 are challenging reactions to perform in chemical catalysis that 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 (a-ketoglutarate) and solvated the structure in a water environment. Thereafter, we set up a series of QM/MM calculations and validated the results against experimental data. Subsequently, we investigated mechanisms leading to C5−and C4−hydroxylation and C4−C5−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 C5−H position and gives a radical intermediate, although the hydrogen atom abstraction from the C4−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 C5 radical intermediate gives bifurcation pathways with a small second hydrogen atom abstraction from the C4−H group and a much higher OH rebound barrier. We also located a charge-transfer intermediate of an iron(II)-hydroxo species with cationic substrate but its kinetics and thermodynamics with respect to the radical intermediate makes 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.
Original languageEnglish
JournalACS Catalysis
DOIs
Publication statusPublished - 1 Aug 2023

Keywords

  • Inorganic reaction mechanisms
  • Computational Chemistry
  • Nonheme iron
  • Dioxygenases
  • Oxygen

Fingerprint

Dive into the research topics of 'How Does the Nonheme Iron Enzyme NapI React Through L-Arginine Desaturation Rather Than Hydroxylation? A QM/MM Study.'. Together they form a unique fingerprint.

Cite this