Why urea hydrolysis costs two Nickels

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Why urea hydrolysis costs two Nickels

• Stefano Benini D. Phil.
• Structural Biology Laboratory
• ELETTRA Synchrotron
• Trieste, Italy

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What is urease?

• Is a protein produced by bacteria fungi and
  plants
• Is a nickel enzyme catalysing the hydrolysis of
  urea
• The first enzyme to be crystallised (Sumner 1926)
• The first protein shown to contain nickel (Dixon
  1975)

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Why is urease important?

• Involved in human and animal diseases
• Involved in nitrogen turnover
• Involved in the rapid hydrolysis of urea in
  agricultural fertilisation
• Urea half life is 3.6 years in water at 38C
• The catalysed reaction is 1014 times faster then
  the uncatalysed one

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Why working on urease from Bacillus pasteurii?

• B. pasteurii is a bacterium living in the soil
  producing large amounts of urease
• The structure of Klebsiella aerogenes urease
  (Jabri et al. 1995) showed an incorrect
  coordination of the nickel ions
• The reaction and the inhibition mechanisms were
  still unclear
• To provide useful information for structure based
  drug design

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K. aerogenes urease native active site (Jabri et
al. 1995)
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Project organisation

• Crystallisation and structure solution of native
  urease from B. pasteurii
• Complexes with several inhibitors belonging to
  different chemical classes
• acetohydroxamic acid, b-mercaptoethanol,
  phosphate, diamidophosphate
• Structural comparison between the native and the
  inhibited structures

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B. pasteurii urease crystals
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B. pasteurii urease diffraction pattern (20-1.65
Å)
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The molecular replacement
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B. pasteurii urease Ramachandran plot
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Ile b-99 is not allowed!
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The carbamylated N-terminus
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Native B. pasteurii urease active site
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Native B. pasteurii urease active site
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The active site hydrogens
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Acetohydroxamic acid inhibited urease
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Acetohydroxamic acid inhibited urease
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b-mercaptoethanol inhibited urease
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b-mercaptoethanol inhibited urease
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b-mercaptoethanol inhibited urease
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Phosphate inhibited urease
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Phosphate inhibited urease
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Diamidophosphate inhibited urease
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Diamidophosphate inhibited urease
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The active site flap
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The reaction mechanism

• Binding of urea
• Nucleophilic attack by the bridging hydroxide
• Protonation of the distal urea nitrogen
• Release of the reaction products and flap opening

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The role of His a323
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But why Nickel?

• Because of the multiple binding sites (protein
  and ligand) required
• Nickel has octahedral coordination, preferred to
  zinc which is mostly tetrahedral
• Nickel has higher affinity to nitrogen-based
  ligands than zinc

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Future work

• Complex with mercaptoethylamine
• Complex with borate
• Inactive mutant with urea bound to the active
  site
• Structure based drug design