1. Uroporphyrinogen Decarboxylase (UROD)
Catie Hattan

2. Picture Ref. 4
In the heme biosynthetic pathway the enzyme uroporphyrinogen decarboxylase’s, UROD, is responsible for the decarboxylation of uroporphyrinogen III, URO III, to the final product coproporphyrinogen III (copro III).1 When a mutation of this enzyme occurs it leads to the accumulation of intermediates and related porphyrins found in porphyrias.

3. Secondary & Quaternary Structure
Secondary Structure
Quaternary Structure
Human vs. Bacillus subtilis
Human UROD
51% helical (21 helices; 188 residues)
10% beta sheet (10 strands; 40 residues)
Bacillus subtilis
50% helical (21 helices; 182 residues)
8% beta sheet (11 strands; 29 residues)
Human vs. Bacillus subtilis
Human UROD
Homodimer
1 Monomer 367 residues
Bacillus subtilis
1 Monomer 359 residues
PDB ID: 1R3Y HUMAN UROD
PDB ID: 2INF BACILLUS SUBTILIS

4. UROD “Clockwise” Decarboxylation
Major Isomers =7d, 6da, 5dab
Isomers=7a, 7b, 7c
Isomers=6ab, 6ac, 6bc, 6bd, 6cd
Mechanism Photo from Reference 1
The route of decarboxylation from UROIII to Copro III produces three intermediates. Of these intermediates 14 different isomers can form.1 The question was raised is there a “preferred” route of decarboxylation under physiological conditions with only a single major isomer of each intermediate present or is the route always random? A study carried out by A. H. Jackson and coworkers poisoned rats with hexachlorobenzene which causes the mice to excrete intermediates similar to people suffering from porphyrias.2 For comparison all 14 intermediates were produced as carboxylate esters and compared against the intermediates isolated from the mice.2 A single isomer each of the hepta-, hexa-, and penta- carboxylate intermediate was confirmed as the major ones present under physiological conditions giving rise to the “clockwise” decarboxylation seen here.2 It was found that when substrate to enzyme ratios were high it seemed for to follow a more random route.5
Isomers=5abc, 5acd, 5bc

5. Understanding the decarboxylation pathway that this enzyme follows is of interest but something also noteworthy about this enzyme is that it requires no cofactors be present to initiate the mechanism. So how does this mechanism work? It has been proposed that the substrate, URO III, acetate side chain and Arg37 residue of the enzyme creates an ionic interaction which they allows the substrate to brought into the non-polar active site of the enzyme. Then the Asp86 present is believed to protonate the alpha position of the pyrrole ring. The Asp 86 then helps to stabilize the species. Once protonated the elimination of CO2 forms an exocyclic double bond. This species then removes a proton from the arg 37 forming a methyl group where the acetate side chain had been. Finish up with reprotonation of Asp86.1,4
P=CH2CH2CO2H

6. Residues Important to Enzyme Activity
BACTERIA1
BACTERIA2
BARLEY
TOBACCO
BACTERIA3
YEAST
RAT
HUMAN
ORANGUTAN
FROG
SALMON
Asp 86
Arg 37
BACTERIA1
BACTERIA2
BARLEY
TOBACCO
BACTERIA3
YEAST
RAT
HUMAN
ORANGUTAN
FROG
SALMON

7. Human UROD Monomer
Alpha Helices=White PDB ID: 1R3Y
Beta Sheets=Blue
Arg 37=Purple
Asp 86=Yellow
Copro III Product=Green

8. PDB ID: 1R3Y
Active Site of Enzyme

9. Bacillus subtilis Homodimer
PDB ID: 2INF

10. Human UROD Monomer in Comparison to Bacillus Subtilis
Arg 37=Green
Arg 41=Red
Asp 86=Blue
Tyr 164=Purple
His 339=Pink
Ile 87=Magenta
Phe 154=Orange
Phe 217=Yellow
Copro III product=Gold
PDB ID: 13RY

11. Bacillus subtilis Monomer UROD in comparison to Human UROD
Arg 29=Green
Arg 33=Red
Asp 78=Blue
Tyr 154=Purple
His 322=Pink
Ile 79=Magenta
Phe 144=Orange
Phe 207=Yellow
PDB ID: 2INF

12. Homodimer of Wild Type Human UROD superimposed with 3 Mutations of F-PCT
Reference 6
The mutation of D306Y is the most significant in this study because they found in this particular mutant that the enzyme becomes insoluble due to a vital hydrogen bond interaction that is comprised. When this hydrogen bond interaction is lost the dimer interface is destabilized.
Yellow Mutation=D306Y
Blue Mutation=G318R
Green Mutation=K297N
Overlay Dimer=G318R Gray
Overlay Dimer=K297N Lime

13. Effects of Mutation in Patients with F-PCT
Reference 6
Kinetic studies comparing the wild type of the Human UROD and two mutant forms of the enzyme found in patients with familial porphyria cutanea tarda.

14. Medical Significance Porphyrias Porphyria Cutanea Tarda
Symptoms & Causes of PCT
Most common type of porphyria.
It can be acquired and it also can be inherited (autosomal dominant).
When exposed to the sun the skin particularly on the face and hands can be damaged.
PCT is caused by an increased concentration of porphyrins in the liver.
PCT can be initiated by iron, alcohol, HCV, HIV, and estrogens.
Reference 7

15. Medical Significance Cont.
How is it diagnosed?
How is it treated?
PCT is diagnosed by determining the amount of porphyrins in the plasma.
Also it can be diagnosed by determining the presence of hepta-carboxylate intermediate in urine.
In feces, isocoproporphyrin, can be isolated to diagnose PCT.
Routine blood draws equal to ~1-2 pints of blood every 1-2 weeks. This is done to reduce the amount of iron in the liver. Target level of ferritin is ~20ng/mL.
And the use of drugs such as chloroquine and hydroxychloroquine help to treat PCT.
Reference 7

16. Questions?

17. PCT Symptoms Blistering occurs when exposed to light.
Areas most effected by sun exposure are the hands and face.

18. References
Lash, T. D.; Mani, U. K.; Lyons, E. A.; Thientanavanich, P.; Jones, M. Normal and Abnormal Heme Biosynthesis. 2. Synthesis and Metabolism of Type-III Pentacarboxylic Porphyrinogens: Further Experimental Evidence for the Enzymatic Clockwise Decarboxylation of Uroporphyrinogen-III. J. Org. Chem. 1999, 64,
2 Jackson, A. H.; Sancovich, H. A.; Ferramola, A. M.; Evans, N.; Games, D. E.; Matlin, S. A.; Elder, G. H.; Smith, S. G. Macrocyclic Intermediates in the Biosynthesis of Porphyrins. Phil. Trans. R. Soc. Lond. B 1976, 273,
Lewis, Jr., C. A., and Wolfenden, R. (2008) Uroporphyrinogen Decarboxylation as a Benchmark for the catalytic proficiency of enzymes. Proc. Natl. Acad. Sci. U. S. A. 105,
Pathway of Heme Biosynthesis. porphyrin.php (accessed Oct 30, 2014).
Jones, R. M., and Jordan, P. M. (1993) Purification and properties of the uroporphyrinogen decarboxylase from Rhodobacter sphaeroides. Biochem. J. 293,
Warby, C. A., and et. al. (2009) Structural and Kinetic Characterization of Mutant Human Uroporphyrinogen Decarboxylases. Cell. Mol. Biol. 55,
Porphyria Cutanea Tarda. (accessed Nov 18, 2014).
Porphyria Cutanea Tarda. (accessed Nov 20, 2014).