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Table 2 Predicted effect of novel missense mutations on FECH structure and function.

From: Loss-of-Function Ferrochelatase and Gain-of-Function Erythroid-Specific 5-Aminolevulinate Synthase Mutations Causing Erythropoietic Protoporphyria and X-Linked Protoporphyria in North American Patients Reveal Novel Mutations and a High Prevalence of X-Linked Protoporphyria

Mutation

SIFTa score/tolerated

PolyPhena score/tolerated

Predicted effect

p.T116P

0.19/Yes

1.000/No

T116 is in a critical position at the end of a small helix that moves to close the active site upon porphyrin binding. Replacement with proline will make this rigid and/or create a kink so that the protein should have subnormal function. Arginine 115, which is important for substrate binding, is located within this helix and its position is likely to be affected by the T116P-induced conformation change.

p.S151F

0.17/Yes

1.000/No

This serine residue is on the backside of the enzyme, away from the active site, and is predicted by SIFT to be tolerated. However, it is located at the end of a helical structure, where it forms a hydrogen bond with threonine 154, stabilizing a tight β turn. The substitution of the bulky hydrophobic benzyl ring of the mutant phenylalanine for the smaller hydrophilic serine might cause protein instability because of misfolding.

p.I206T

0.00/No

1.000/No

This is an internal residue that is in the middle of a helix. The mutation would disrupt local structure and alter overall tertiary structure.

p.R215W

0.19/Yes

0.985/No

This is a surface residue located on the side of the protein. It is not clear how it would affect the structure of the protein, but if this region is involved in protein-protein interactions, substitution of a large bulky residue for a charged residue should have an impact on such an association.

p.M219R

0.01/No

0.998/No

This residue is at the end of a β sheet and would cause structural perturbations because of the addition of the bulky guanido moiety with its positive charge.

p.L265R

0.00/No

1.000/No

L265 is located adjacent to the 2-vinyl group in the substrate-bound structures, and substitution of an arginine would be expected to have a significant negative impact on macrocycle binding in the active site.

p.C411F/Y

0.01/No

1.000/No

The 411F and Y mutation would not have the (2Fe-2S) cluster required for enzyme activity, since C411 is one of the four essential ligands.

  1. aSIFT and PolyPhen are in silico phenotyping tools, as described in Materials and Methods.