L-ornithine N5 monooxygenase is classified under two EC numbers - EC1.14.13.195[1] and EC 1.14.13.196.[2] The first number, 1, identifies the enzyme as an oxidoreductase.[1][2] The subsequent 14 refers to the fact that this enzyme acts "on paired donors, with incorporation or reduction of molecular oxygen".[1][2] The 13 identifies this enzyme as using NADH or NAD(P)H as one donor, while incorporating one atom of oxygen onto the other.[1][2] This is why there are two EC numbers for this enzyme - one ends with 195 referring to NADPH as the donor, while the 196 refers to NAD(P)H as the donor.[1][2]
Crystallographic structures have been solved for this class of enzymes from Aspergillus fumigatus.[7] These structures reflect structural changes which take place when the enzyme binds combinations of ligands, including ornithine and NADP.[7] Additional structures have also been solved for strain Af293.[8][9] These structures reflect different redox and ligation states.[5][8][9] The following table briefly describes these crystal structures:
N-hydroxylating flavin-containing monooxygenase (NMO) enzymes such as this target the nucleophilic terminal amine groups of primary aliphatic amines such as L-ornithine.[7] The enzyme operates via a multistep oxidative mechanism which has a C4a-hydroperoxyflavin intermediate.[5] SidA stabilizes this intermediate and keeps NADP+ bound throughout the remainder of the catalytic cycle because it is necessary for intermediate stabilization.[7] The nicotinamide-ribose moiety and H-bonding between the main chain and residues Lys107, Asn293, and Ser469 position the L-ornithine alpha carbon such that its side chain amino group can be hydroxylated by the C4a-(hydro)peroxyflavin.[7] Unlike many other NMOs, A. fumigatus SidA strictly acts on ornithine.[7] Interactions with arginine increase interactivity between the reduced flavin and oxygen.[7]
The active site is located within a cleft at the interface between the three domains on each subunit. SidA has a resting state (6X0H) in which neither L-ornithine nor NAD(P)H is bound.[5] This resting state has an "out" active site caused by large rotations of the FAD isoalloxazine and a 10-Å movement of the Tyrosine loop.[5] Either flavin reduction or NAD(P)H binding drives the active site to the "in" conformation (6X0I).[5]
SidA demonstrates typical kinetics when saturated with L-ornithine.[10] Inhibition is caused by high concentrations of NADPH and NADH.[10] There is an 8-fold increase in catalytic efficiency for NADPH compared to NADH.[10] NADP+ is a competitive inhibitor with respect to NADPH.[10]
In A. fumigatus, it is classified as a flavoprotein because FAD is a cofactor. It catalyzes the FAD and NADPH-dependent hydroxylation of L-ornithine in biosynthesis of the ferrichromesiderophores triacetylfusarinine and desferriferricrocin.[3][4][10] It is produced primarily under iron-limited conditions.[10] Siderophores are also important for virulence.[7]
In Kutzneria sp. 744, this enzyme is involved in the biosynthesis of piperazate, which contributes to the biosynthesis of kutzneride antifungalantibiotics.[3][4]
References
^ abcdefg"EC 1.14.13.195". IUBMB Enzyme Nomenclature. International Union of Biochemistry and Molecular Biology.
^ abcdefg"EC 1.14.13.196". IUBMB Enzyme Nomenclature. International Union of Biochemistry and Molecular Biology.
^ abcd"EC 1.14.13.195". KEGG Enzyme. Kyoto Encyclopedia of Genes and Genomes (KEGG). Retrieved 2022-10-22.
^ abcd"EC 1.14.13.195". KEGG Enzyme. Kyoto Encyclopedia of Genes and Genomes (KEGG). Retrieved 2022-10-22.