Human uroporphyrinogen III synthase: NMR-based mapping of the active site

Luis Cunha, Miklos Kuti, David F. Bishop, Mihaly Mezei, Lei Zeng, Ming Ming Zhou, Robert J. Desnick

Research output: Contribution to journalArticlepeer-review

19 Scopus citations


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 13C α (100%), 1Hα (99.6%), and nonproline 1HN and 15N resonances (94%) was achieved as well as 85% of the side-chain 13C and 1H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors ND-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 15N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex.

Original languageEnglish
Pages (from-to)855-873
Number of pages19
JournalProteins: Structure, Function and Bioinformatics
Issue number2
StatePublished - 1 May 2008


  • Chemical shift perturbation
  • Enzyme complex
  • Heme biosynthesis
  • Molecular docking
  • Porphyria
  • Resonance assignments


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