Abstract
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. 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 (k(cat) /K(m) ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k(cat) ) and Michaelis constant (K(m) ) of Nii4-WT were lower than those of Nii3-WT. However, the k(cat) /K(m) of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k(cat) /K(m) was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.
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Claims 8
Claim · model-written summary
Nii3 and Nii4 had very similar crystal structures.
- Taxon
- tobacco
Nicotiana tabacumt0 - Gene
- Nii3 and Nii4e0
- Outcome
- very similar structurese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
t0“tobacco”
Claim · model-written summary
The Nii3 structure resolved geometries of the iron sulfur cluster and siroheme groups.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Nii3e0
- Outcome
- detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groupse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups”
t0“tobacco”
Claim · model-written summary
The hydrophobic Met175 side chain facilitated Nii3 enzyme efficiency.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Met175e0
- Outcome
- enzyme efficiencye0
- Direction
- facilitatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k(cat) /K(m) )”
t0“tobacco”
Claim · model-written summary
Nii4 wild type had a lower turnover number than Nii3 wild type.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Nii4-WTe0
- Outcome
- turnover number (k(cat) )e0
- Direction
- lowere0
- Comparator
- Nii3-WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the turnover number (k(cat) ) and Michaelis constant (K(m) ) of Nii4-WT were lower than those of Nii3-WT”
t0“tobacco”
Claim · model-written summary
Nii4 wild type had a lower Michaelis constant than Nii3 wild type.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Nii4-WTe0
- Outcome
- Michaelis constant (K(m) )e0
- Direction
- lowere0
- Comparator
- Nii3-WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the turnover number (k(cat) ) and Michaelis constant (K(m) ) of Nii4-WT were lower than those of Nii3-WT”
t0“tobacco”
Claim · model-written summary
Nii4 wild type catalytic efficiency was about 1.4 times that of Nii3 wild type.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Nii4-WTe0
- Outcome
- k(cat) /K(m)e0
- Direction
- highere0
- Value
- 1.4 timese0
- Comparator
- Nii3-WTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the k(cat) /K(m) of Nii4-WT was about 1.4 times higher than that of Nii3-WT”
t0“tobacco”
Claim · model-written summary
Residue 448 difference accounted for the catalytic efficiency difference in variants.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Residue 448e0
- Outcome
- change in k(cat) /K(m)e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the change in k(cat) /K(m) was brought about by the difference in Residue 448”
t0“tobacco”
Claim · model-written summary
Authors propose Nii3 has low affinity and Nii4 high affinity.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- Nii3e0
- Outcome
- Nii3 is the low-affinity and Nii4 is the high-affinity aNiRe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR”
t0“tobacco”