Structural and functional studies of Assimilatory Nitrite Reductase

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3) and root-type (Nii2 and Nii4). The high resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions respectively, revealed that both proteins had very similar structures. Although these structures are almost identical to spinach aNiR that were previously obtained at 2.8 Å resolution by Knaff & Allen’s group, the Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (kcat/Km). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (kcat) and Michaelis constant (Km) of Nii4-WT were lower than those of Nii3-WT. By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.