Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site

Martin A. Walsh, Zbyszek Otwinowski, Anatassis Perrakis, Paul M. Anderson, Andrzej Joachimiak

Research output: Contribution to journalArticle

46 Citations (Scopus)

Abstract

Background: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. Results: We have determined the crystal structure of cyanase at 1.65 resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. Conclusions: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.

Original languageEnglish (US)
Pages (from-to)505-514
Number of pages10
JournalStructure
Volume8
Issue number5
DOIs
StatePublished - May 1 2000

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Catalytic Domain
Enzymes
Cyanates
Selenomethionine
Amino Acid Sequence Homology
Proteins
Oxalates
Enzyme Inhibitors
Operon
Bicarbonates
Selenium
cyanate hydrolase
Ammonia
Carbon Dioxide
Anions
Chlorides
Binding Sites
Escherichia coli
Bacteria
DNA

Keywords

  • Active site
  • Cyanase
  • Decamer structure
  • MAD phasing
  • Monoanion and dianion inhibitors
  • Synchrotron radiation

ASJC Scopus subject areas

  • Molecular Biology
  • Structural Biology

Cite this

Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site. / Walsh, Martin A.; Otwinowski, Zbyszek; Perrakis, Anatassis; Anderson, Paul M.; Joachimiak, Andrzej.

In: Structure, Vol. 8, No. 5, 01.05.2000, p. 505-514.

Research output: Contribution to journalArticle

Walsh, Martin A. ; Otwinowski, Zbyszek ; Perrakis, Anatassis ; Anderson, Paul M. ; Joachimiak, Andrzej. / Structure of cyanase reveals that a novel dimeric and decameric arrangement of subunits is required for formation of the enzyme active site. In: Structure. 2000 ; Vol. 8, No. 5. pp. 505-514.
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N2 - Background: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. Results: We have determined the crystal structure of cyanase at 1.65 resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. Conclusions: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.

AB - Background: Cyanase is an enzyme found in bacteria and plants that catalyzes the reaction of cyanate with bicarbonate to produce ammonia and carbon dioxide. In Escherichia coli, cyanase is induced from the cyn operon in response to extracellular cyanate. The enzyme is functionally active as a homodecamer of 17 kDa subunits, and displays half-site binding of substrates or substrate analogs. The enzyme shows no significant amino acid sequence homology with other proteins. Results: We have determined the crystal structure of cyanase at 1.65 resolution using the multiwavelength anomalous diffraction (MAD) method. Cyanase crystals are triclinic and contain one homodecamer in the asymmetric unit. Selenomethionine-labeled protein offers 40 selenium atoms for use in phasing. Structures of cyanase with bound chloride or oxalate anions, inhibitors of the enzyme, allowed identification of the active site. Conclusions: The cyanase monomer is composed of two domains. The N-terminal domain shows structural similarity to the DNA-binding α-helix bundle motif. The C-terminal domain has an 'open fold' with no structural homology to other proteins. The subunits of cyanase are arranged in a novel manner both at the dimer and decamer level. The dimer structure reveals the C-terminal domains to be intertwined, and the decamer is formed by a pentamer of these dimers. The active site of the enzyme is located between dimers and is comprised of residues from four adjacent subunits of the homodecamer. The structural data allow a conceivable reaction mechanism to be proposed.

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