Sandbox Reserved 1677
From Proteopedia
(Difference between revisions)
| Line 29: | Line 29: | ||
<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. |
| + | |||
| + | The article mentions that the unambigous electron densities for NAD+ and octanal define how lingands bind to the AldC C291A mutant and is the one that indicates the location of the substrate and cofactor-binding sites. | ||
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 | ||
| Line 41: | Line 43: | ||
The N-terminus of Aldc contains a central beta sheet surrounded by alpha helices which forms the NAD(H)-binding site. | The N-terminus of Aldc contains a central beta sheet surrounded by alpha helices which forms the NAD(H)-binding site. | ||
Additionally, around the C-terminus there is a mixture of alpha and beta domains which includes the cysteine residue and forms the aldehyde binding site. A small three stranded beta sheet domain facilitates aligomerization. There is an interdomain linker region that connects the N and C terminal domains of Aldc. | Additionally, around the C-terminus there is a mixture of alpha and beta domains which includes the cysteine residue and forms the aldehyde binding site. A small three stranded beta sheet domain facilitates aligomerization. There is an interdomain linker region that connects the N and C terminal domains of Aldc. | ||
| + | |||
| + | The Amino acid sequence highlight how the catalytic cysteine and residues of the NAD(H)-binding site are highly conserved with major variations in the substrate binding site which leads to functional differences in Aldehyde dehydrogenases. Amino acids in AldC in the oligomerization domain change the electrostatic surface charge and the surface topology. | ||
| Line 46: | Line 50: | ||
=== 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. | 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 | Cysteine is the most important catalytic residues | ||
Revision as of 15:35, 25 April 2021
| |||||||||||
