Sandbox Reserved 1677
From Proteopedia
(Difference between revisions)
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<b> Trp 160 Tyr 163, Trp 450, Phe 456, Tyr 458, met 114, leu 118 </b> | <b> Trp 160 Tyr 163, Trp 450, Phe 456, Tyr 458, met 114, leu 118 </b> | ||
Apolar interactions dominate the octanal binding in the hydrophobic substrate binding pocket. A cluster of aromatic residues and two nonpolar residues (Methionine and Leucine) peovides hydophobic environment that accommodates octanal and other aliphatic aldehydes. The substrate binding site forms an aromatic box for adaptable apolar ligand interaction. | Apolar interactions dominate the octanal binding in the hydrophobic substrate binding pocket. A cluster of aromatic residues and two nonpolar residues (Methionine and Leucine) peovides hydophobic environment that accommodates octanal and other aliphatic aldehydes. The substrate binding site forms an aromatic box for adaptable apolar ligand interaction. | ||
| - | To examine the contribution of the active site residues, a series of site directed mutants targeting residues in the NAD(H) binding site and the octanal binding site were generated. All the 31 mutants were expressed in E.coli and purified using nickel-affinity and size exclusion chromatographies. The enzyme activity screening showed that mutation of the catalytic residues in the NAD(H) binding site and octanal binding site resulted in enzyme with less than 1% of WT specific activity. | + | To examine the contribution of the active site residues, a series of site directed mutants targeting residues in the NAD(H) binding site and the octanal binding site were generated. All the 31 mutants were expressed in E.coli and purified using nickel-affinity and size exclusion chromatographies. The enzyme activity screening showed that mutation of the catalytic residues in the NAD(H) binding site and octanal binding site resulted in enzyme with less than 1% of WT specific activity. |
https://proteopedia.org/wiki/images/4/48/Screen_Shot_2021-04-18_at_4.03.57_PM.png | https://proteopedia.org/wiki/images/4/48/Screen_Shot_2021-04-18_at_4.03.57_PM.png | ||
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=== Other important features === | === Other important features === | ||
<scene name='87/873239/Rossmann_fold_motifs/1'>The Rossmann fol</scene>d of the NAD(H) binding domain provides extensive polar and apolar interactions that position the nicotinamide ring of NAD+ in proximity to the C291A point mutation. Its main function is to bind NAD+ cofactor and contribute to substrate binding. The cleft found in the protein is where the active site is, the binding cleft contains the octanal binding amino acids | <scene name='87/873239/Rossmann_fold_motifs/1'>The Rossmann fol</scene>d of the NAD(H) binding domain provides extensive polar and apolar interactions that position the nicotinamide ring of NAD+ in proximity to the C291A point mutation. Its main function is to bind NAD+ cofactor and contribute to substrate binding. The cleft found in the protein is where the active site is, the binding cleft contains the octanal binding amino acids | ||
| + | Glu 391 Mutation, loss of Glu 257 side chain removed the internal interaction that helps position the amine group of the nicotinamide and changes at this residue could change the orientation of the nicotinamide group for hydride transfer. | ||
| + | Cysteine is the most important catalytic residues | ||
<scene name='87/873239/Cartoon_view/5'>Interactions</scene> between amino acids. This helps to understand how the polar amino acids | <scene name='87/873239/Cartoon_view/5'>Interactions</scene> between amino acids. This helps to understand how the polar amino acids | ||
Revision as of 15:07, 19 April 2021
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