Temp model of POR
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Description
POR R498PNADPH--cytochrome P450 reductase Proline is the worse residue for a α-helix. The structure of an α-helix is thanks to the hydrogen bonding between the hydrogen of the backbone-nitrogen and the backbone ketone of the residue 3-before whereas in a proline the Cδ takes the place of the hydrogen. A prior a R to P mutation results in an increase of 3 kcal/mol. But it also shifts apart the residues, in this case the C-terminus of the helix is strongly bonded by N292, while the N-terminus (T495) coordinates the phosphate of the FAD ligand. In the following image, the mutation was added and the structure relaxed (energy minimisation) and opposed to being rebuild for the nearby residues, therefore the structure tries to be helical still.The energy difference is not major, but the shift could strongly affect the reaction of FAD.The arginine side chain is free. The missing density in the subsequent loop was solved, but it did not influence the arginine.The pI of the protein is 5.28 (Expasy protparams), but the face where the arginine is quite positive. https://www.sciencedirect.com/science/article/pii/S0002929707607216?via%3Dihub How do Huang et al quantify activity?They express the protein in E. coli (while keeping the N-terminus by making sphaeroplasts).They do two assays (and calculate full Michaelis-Menten kinetics):· Cytochrome c reduction of NADH (regenerated via GDH) monitored at 550 nm· Cytochrome P450c17 17α-hydroxylase reaction on progesterone measured via chromatography (TLC)· Cytochrome P450c17 17,20 lyase reaction on 17α-hydroxypregnenolone### peptide and nucleic acid chains
- NADPH--cytochrome P450 reductase [offset by -3 (:A)]
Changes
Chains removed. Dehydrated.