Alexandra Young CHE 442 Uroporphyrinogen Decarboxylase (URO-D)

sigmaaldrich.com Heme and Uroporphyrinogen Decarboxylase (URO-D)  5 th enzyme of the heme biosynthetic pathway  Responsible for catalyzing the conversion of uroporphyrinogen III to coproporphyrinogen III  Decarboxylates the acetate side chains of uroporphyrinogen, converting them into methyl substituents  Functions as a homodimer in solution  Regarded as an unusual decarboxylase because it does not use the assistance of cofactors  Deficiency or mutations in the URO-D enzyme are known to result in porphyria *At low substrate concentrations the reaction is believed to follow an ordered route, with the sequential removal of CO 2 from the D, A, B, then C ring, whereas at higher substrate/enzyme levels there appears to be no order

Uroporphyrinogen Decarboxylase Deficiency: Porphyria Deficiency and/or mutations in the URO-D enzyme can result in porphyria. Porphyria is a disorder that causes the overproduction and accumulation of porphyrins (or their chemical precursors) in tissue and is marked with either neurological complications or skin problems. Hepatic porphyria: seizures, psychosis, extreme back and abdominal pain and acute polyneuropathy Erythropoietic porphyrua: light-sensitivity, disfiguration, blistering rash, increased hair growth. *Based on some similarities between these conditions and popular folklore, porphyria has been suggested as a possible explanation for the origin of vampire and werewolf legends.

Heme Biosynthesis and URO-D Substituent Key: Pr = Propionate -CH 2 CH 2 CO 2 - Ac = Acetate -CH 2 CO 2 - Vi = Vinyl -CH=CH 2 URO-D

The 5 th Step in Heme Synthesis Uroporphyrinogen Decarboxylase: UROD UROD The mechanism of URO-D has been proposed to proceed through coordination by an aspartate residue and protonation of the substrate by an arginine residue in the active site cleft. J. Phys. Chem. B, Vol. 109, No. 38, 2005 Uroporphyrinogen III Coproporphyrinogen III

URO-D AA Sequence and Structure 1 meanglgpqg fpelkndtfl raawgeetdy tpvwcmrqag rylpefretr aaqdffstcr 61 speacceltl qplrrfplda aiifsdilvv pqalgmevtm vpgkgpsfpe plreeqdler 121 lrdpevvase lgyvfqaitl trqrlagrvp ligfagapwt lmtymveggg sstmaqakrw 181 lyqrpqashq llriltdalv pylvgqvvag aqalqlfesh aghlgpqlfn kfalpyirdv 241 akqvkarlre aglapvpmii fakdghfale elaqagyevv gldwtvapkk arecvgktvt 301 lqgnldpcal yaseeeigql vkqmlddfgp hryianlghg lypdmdpehv gafvdavhkh 361 srllrqn The EMBO Journal Vol.17 No.9 pp.2463–2471, 1998

Multiple Sequence Alignment (There are many invariant or highly conserved residues, however, only a select few with designated properties have been highlighted) Key: Color Coded by Proposed Functionality of Residue Active Site Cleft……………...……Arg37,Arg41,Arg50,etc Catalysis…………………..…......…Asp86,Tyr164 Hydrogen Bonding…………..…….Gln38,Gln302,Asn336 Hydrophobic Core…………..……..Ala22,Pro32,Trp34,Leu91,Leu337 Substrate Binding…….……………Solvent Exposed Hydrophobic Residues Hydrogen Bonding to Substrate……Ser219

URO-D Structure Uroporphyrinogen Decarboxylasae (URO-D)  40.8 kDa and 367 AA residues  Single domain but forms a homodimer in solution  Many conserved residues in active site cleft  Contains a (β/α) 8 -Barrel  Deep active site cleft formed by loops at C- terminus of barrel strands

URO-D Complexed with Product Coproporphyrinogen III Asp 86 Arg 37 Asp 86 Leu 88 Coordinating InteractionsMechanistic Interactions Coproporphyrinogen III Product  Asp86 is the only negatively charged AA in the active site  The pyrrole units are angled downward for H-bond interactions with Asp86  Precise orientation of the product is held by H-bonding between Leu88 and Asp86  Protonation of the substrate by Arg37 initiates the decarboxylatioin mechanism

Role of Asp86 in Binding and Catalysis Structure and Activity of Asp86 Mutants  Asp86Gly (-/neut) Coordination of 2 H 2 O replace Asp for similar substrate geometry, but very low activity, so Asp86 plays a direct role in catalysis  Asp86Asn (-/+) Inability to bind substrate, repulsive interaction with pyrrole NHs, very low activity  Asp86Glu (-/-) Same H-bond interaction with substrate, but reduced activity, so not providing all interactions necessary for optimal catalysis: reduced contact with surrounding hydrophobic residues, distortion of sites required for protonation of the pyrroles or decarboxylation of the acetates

Active Site Cleft  Extensive interactions are formed between one face of the substrate and a ring of invariant or conserved hydrophobic residues  Ser219 projects into the active site cleft and may H-bond to the substrate  There are 10 solvent exposed hydrophobic side chains that may bind to substrate (Met36, Phe46, Phe55, Ile82, Phe84, Ile87, Leu88, Phe154, Phe217, Phe261)  Binding of the substrate acetate group in a hydrophobic environment at the bottom of the active site cleft may contribute to catalysis by destabilization of the charged substrate with respect to the carbon dioxide product.

URO-D Homo-Dimer  Dimerizes in solution (K d = 0.1 μM )  Assembly of the dimer places active site clefts in juxteposition  Possibility of functionally important interaction between the catalytic centers (Unclear if the dimer forms one large active site and binds only one substrate or combines two smaller active sites)  Dimer interface is the Helix formed by loops L3, L4, L5, L6, L7, L8  Interface formed by loops is flat and has a total of 2387 Å 2 of solvent-accessible surface area that is buried upon dimerization  Interface is largely hydrophilic and buries 27 ordered H 2 O molecules (Inculdes loops composed of AA: L3=gggstm- L4=shaghlgpqlfn kfalp- L5=kdg- L6=agagy- L7=dwtva- L8=lghglypdmd)  Dimerization forms a deeper cleft that is more protected from solvent Interface of Interaction View from the center of the dimer, looking down the interface Interacting loops

(β/α) 8 - Barrel Asp 86 Arg 37 Leu 88  Predominately hydrophobic  Central residues pack in 3-4 layers  Formed by loops L1-L4 and L8  Forms a deep active site cleft  15 X 15 X 7 Å  C-terminal ends of the loops

Kinetics Uroporphyrinogen III to Coproporphyrinogen III  [S] = μM  Km = 1.8 μM  Substrate inhibition at high concentrations of URO decreases activity of URO-D and decarboxylation proceeds in a random fashion (a) The effect of substrate concentration on URO-D activity (b) The effect of uroporphyrin I and III on URO-D activity (c) The optimal pH of URO-D (uroporphyrinogen III in phosphate buffer) (d) The effect of prolonged incubation at 55 ˚C

Materials and Methods Protein Chemistry: Recombinant histidine-tagged human URO-D Over expressed in E.coli Purified with nickel-chelate column chromatography Crystalization: Crystallized in sitting drops at 4˚C in citrate buffer 5 μl URO-D solution (6 mg/ml) 50 mM Tris-HCl pH 7.5 1mM βMe 10% glycerol 2μl reservoir solution containing M citrate pH X-ray Data Collection: 1.60 Å resolution Crystals at 100 K Fuji image plates and an off line scanner were used Structure Determination and Refinement: Contains 267 H 2 O molecules 355 of the 367 residues were refined 31 N-terminal residues and 2 C-terminal residues were disordered

References 1.) Whitby, F.G.; Phillops, J.D.; Kushner, J.P.; Hill, C.P. J. EMBO 1998, 17, ) Silva, P.J.; Ramos, M.J. J. Phys. Chem. B. 2005, 109, ) Phillips, J.D.; Whitby, F.G.; Kushner, J.P.; Hill, C.P. J. EMBO 2003, 23, ) Lash, T.D.; Mani, U.N.; Lyons, E.A.; Thientanavanich, P.; Jones, M.A. J. Org. Chem. 1999, 64, ) Jones, R.M.; Jordan, P.M. Biochem. J. 1993, 203, 703.