title: The mononuclear metal center of type-I dihydroorotase from Aquifex aeolicus.
abstract: BACKGROUND
  Dihydroorotase (DHO) is a zinc metalloenzyme, although the number of active site zinc ions has been controversial. E. coli DHO was initially thought to have a mononuclear metal center, but the subsequent X-ray structure clearly showed two zinc ions, α and β, at the catalytic site. Aquifex aeolicus DHO, is a dodecamer comprised of six DHO and six aspartate transcarbamoylase (ATC) subunits. The isolated DHO monomer, which lacks catalytic activity, has an intact α-site and conserved β-site ligands, but the geometry of the second metal binding site is completely disrupted. However, the putative β-site is restored when the complex with ATC is formed and DHO activity is regained. Nevertheless, the X-ray structure of the complex revealed a single zinc ion at the active site. The structure of DHO from the pathogenic organism, S. aureus showed that it also has a single active site metal ion.
  
  
  RESULTS
  Zinc analysis showed that the enzyme has one zinc/DHO subunit and the addition of excess metal ion did not stimulate catalytic activity, nor alter the kinetic parameters. The metal free apoenzyme was inactive, but the full activity was restored upon the addition of one equivalent of Zn2+ or Co2+. Moreover, deletion of the β-site by replacing the His180 and His232 with alanine had no effect on catalysis in the presence or absence of excess zinc. The 2.2 Å structure of the double mutant confirmed that the β-site was eliminated but that the active site remained otherwise intact.
  
  
  CONCLUSIONS
  Thus, kinetically competent A. aeolicus DHO has a mononuclear metal center. In contrast, elimination of the putative second metal binding site in amidohydrolyases with a binuclear metal center, resulted in the abolition of catalytic activity. The number of active site metal ions may be a consideration in the design of inhibitors that selectively target either the mononuclear or binuclear enzymes.
authors: Brian F P
authors: BF
authors: Edwards
authors: Roshini
authors: R
authors: Fernando
authors: Philip D
authors: PD
authors: Martin
authors: Edward
authors: E
authors: Grimley
authors: Melissa
authors: M
authors: Cordes
authors: Asmita
authors: A
authors: Vaishnav
authors: Joseph S
authors: JS
authors: Brunzelle
authors: Hedeel Guy
authors: HG
authors: Evans
authors: Department of Biochemistry and Molecular Biology, Wayne State University School of Medicine, 540 East Canfield Street, Detroit, MI 48201, USA. drevans@med.wayne.edu.
authors: David R
authors: DR
authors: Evans
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D018360:Crystallography, X-Ray; D004080:Dihydroorotase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006090:Gram-Negative Bacteria / Q000201:enzymology*; D007477:Ions / Q000737:chemistry; D007700:Kinetics; D008670:Metals / Q000737:chemistry* / Q000378:metabolism; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D014867:Water / Q000737:chemistry; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
chemical_list: D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D014867:Water; D003035:Cobalt; D004080:Dihydroorotase; D015032:Zinc