Human uroporphyrinogen III synthase: NMR-based mapping of the active site.
Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.
Cunha
Luis
L
Department of Genetics and Genomic Sciences, Mount Sinai School of Medicine, New York 10029-6574, USA.
Kuti
Miklos
M
Bishop
David F
DF
Mezei
Mihaly
M
Zeng
Lei
L
Zhou
Ming-Ming
MM
Desnick
Robert J
RJ
D000595:Amino Acid Sequence; D001665:Binding Sites; D002247:Carbon Isotopes; D003198:Computer Simulation; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000302:isolation & purification; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D013816:Thermodynamics; D014575:Uroporphyrinogen Decarboxylase / Q000302:isolation & purification; D014576:Uroporphyrinogen III Synthetase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification; D014577:Uroporphyrinogens / Q000494:pharmacology
D002247:Carbon Isotopes; C528490:N(D)-methyl-1-formylbilane; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D014577:Uroporphyrinogens; D011163:Hydroxymethylbilane Synthase; D014575:Uroporphyrinogen Decarboxylase; D014576:Uroporphyrinogen III Synthetase