Group H Presentation: The Mysteries of Urease

1
Group H Presentation The Mysteries of Urease

• Judy Oh
• Michelle Wu
• Ming-Hsiang Hsia
• Michael Galustyan
• Ladan Sahabi

2
Background and Overview

• Urease
• Was first crystallized in 1926 by James Summer
• Function catalyzes the conversion of urea to
  ammonia and carbamate
• Can be found in different types of bacteria and
  other organisms (e.g. Jack bean Urease (JBU),
  Klebsiella aerogeouss Urease, and Helicobacter
  pyloris Urease

3
Urea and Urea Cycle

• Urea is synthesized
• in liver by action of 5
• enzymes
• Less toxic than
• ammonia (ureotelic
• organisms)

4
Reaction of Urease

• Urease produces ammonia and carbamate
• Carbamate is not stable and spontaneously
  decomposes to ammonia and bicarbonate

5
Biological Implications

• Ureases reaction causes increase in pH both in
  animals organism and soil
• Some bacteria (H. pylorii) have this enzyme and
  its action involves in different types of
  diseases hepatic coma and urinary tract
  infections
• Increase in pH is very toxic for plants and lead
  to large agriculture loss
• Future studies will focus on designing of urease
  inhibitors
• We need to understand both structure and
  mechanism of urease in order to create
  inhibitors

6
The Structure of Urease

• Heteropolymeric metalloprotein
• Three independent active sites
• Dinuclear Ni2 centers (3.7Å apart)
• Bridged by carboxylate group of carbamylated Lys
  220
• Ni1 His 249, His 275
• Ni2 His 137, His 139, Asp 363
• Three water hydroxide directly bound to Ni1 and
  Ni2

7
Structure Native Form

• WB, W1, W2, and W3
• WB symmetrically bridges Ni1 and Ni2.
• W1 hydrogen bond with His 222
• W2 hydrogen bond with Ala 170
• Fourth water molecule, W3, interacts with WB, W1,
  and W2
• Asp 363 is at hydrogen bonding distance from WB
  and W3
• Inhibitor complex with lability of water
  molecules and bridging hydroxide, with protein
  framework rigidly maintained

8
Structure, Native Form
9
Structure DAP Inhibited Urease

• Crystallized with Phenylphosphodiamidate (PPD)
• Presence of DAP molecule (Diamidophosphoric acid)
• Replaces the four water molecules
• Bound to Ni1 and Ni2 bia three of four potential
  donor atoms

10
Structure, DAP Inhibited

• Hydrogen binding pattern stabilizes and orients
  inhibitor
• 1) Ni1-bound O H-bonds with His 222
• 2) Ni2-bound DAP amido N H-bonds with Ala170 and
  Ala366
• 3) Asp 363 at H-bond distance from Ni-bridging
  DAP O and distal DAP N
• 4) Distal N of DAP H-bonds with Ala 366 and His
  323
• His 323 located on a helix-loop-helix motif
  capable of both open and closed conformations

11
Structure, DAP inhibited
12
Structure, DAP Inhibited

• Red Native conformation (open)
• Yellow DAP Inhibited conformation (closed)

13
Structure, Boric Acid Inhibition

• Boric Acid is a substrate analogue
• Bridging binding model
• Replaces three water molecules, leaving the
  bridging WB intact
• Ni-Ni distance unperturbed
• Ni1-bound O H-bonds with His 222 N2-bound O
  H-bonds with Ala 366 (same as in the native
  structure)

14
Structure, Boric Acid Inhibition

• Distal B-OH bond H-bonded to a water molecule in
  hydrogen-bonding network with solvent molecule
  Ala366
• Neighboring residues stabilizing B(OH)3 binding
  to dinuclear Ni center
• Lack of reactivity of B(OH)3 with WB could be due
  to unfavorable symmetry and energy of HOMO of WB
  and LUMO of boric acid

15
Structure, Boric Acid Inhibition
16
Mechanisms of Urease

• Two mechanisms of urease in urea hydrolysis
• A) involves Ni-bridging OH as nucleophile
• B) involves terminal OH as nucleophile
• In General
• Substrate is urea
• Urea replaces W1, W2, and W3 positions in active
  site cavity
• Two Ni2 play essential roles
• Tetrahedral transition state
• Products are carbamate and ammonia

17
Mechanisms of Urease
18
Mechanism A of Urease

• Direct Role of Both Ni2 ions in
• Binding and activating substrate
• Urea is a poor chelating ligand, however
• Low Lewis base character of its NH2 groups
• However, formation of strong H-bonds with nearby
  carbonyl oxygen enhance the basicity of NH2 group
• This facilitates the interaction of the amide
  Nitrogen with Ni2 (donate electron pair to Ni2)
• This brings urea carbon in close proximity to the
  Ni-bridging OH anion.

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Mechanism A of Urease

• After the transition state is formed, cleavage of
  ammonia occurs
• Nickel-bridging hydroxide transfers a proton to
  Aspa363 Od2
• Asp a363 Od2 provides proton to distal NH2 to
  form NH3
• Hisa323 (in its deprotonated form) acts as a
  general base, moves in (open to closed
  conformational change) to stabilize the positive
  charge of NH3

Transition State
20
Mechanism A of Urease
21
Mechanism B of Urease

• Problems with Mechanism B
• Missing general base that would deprotonate the
  Ni2 bound water molecule at optimum pH for enzyme
  activity (pH8)
• Role of Hisa323 as a general acid, which must be
  protonated at pH 8 even though it has a pKa of
  6.5
• Therefore, according to this mechanism, only 0.3
  of all urease molecules would be in the optimal
  protonation state for catalysis

2
Note Ni2 bound water needs to be deprotonated
to become a nucleophile OH ion.
22
Urease Competitive Inhibitors

• Urease inhibitors could be used to overcome
  negative side effects caused by the abrupt
  overall pH increase during the enzymatic
  hydrolysis of urea.
• Several classes of molecules have been discovered
  such as diphenols, quinones, hydroxamic acids,
  phosphoramides, and thiols.
• Two examples are ß-mercaptoethanol (BME) and
  acetohydroxamic acid (AHA

23
Structure of BPU inhibited with ß-mercaptoethanol

• Sulfur atom of BME symmetrically bridges the
  binuclear NiCenter
• The inhibitor chelates Ni1 using its terminal OH
  group
• Both Ni ions are pentacoordinated Ni1 is
  distorted square pyramidal, Ni2 is distorted
  trigonal bipyramidal
• A second molecule of BME is involved in disulfide
  bond with Cys a322 and in a H-bond and the
  carbonyl oxygen atom of Ala a366
• Inhibition with BME occurs by targeting enzyme
  sites that both directly and indirectly
  participate in substrate positioning and
  activation

24
Structure of BPU inhibited with acetohydroxamic
acid

• One AHA molecule provides a symmetric bridge
  between Ni1 and Ni2 through the hydroxamate
  oxygen (OB).
• A second AHA oxygen (OT) is bound to Ni1.
• Both Ni ions are trigonal bipyramidal
• The Ni1-Ni2 distance is 3.5 Å.
• The Aspa363 carboxylate group is rotated about
  the Cß-C? bond by 35o with respect to its
  conformation in native BPU, to receive a hydrogen
  bond from the AHA-NH group.
• The AHA molecule behaves as a bridging-chelating
  ligand.

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In vivo Urease Activation and Nickel Trafficking

• Biology of transport and insertion of Ni2 into
  the active site of urease has been extensively
  studied for K. aerogenes
• Key Players
• 1) KaUreD (30D)
• maintains proper conformation of apo-urease by
  forming a specific complex
• 2) KaUreF (25D)
• binds to KaUreD-apo-urease complex to facilitate
  carbamylation of Ni-bridging Lys residue in
  active site by preventing Ni2 ions from binding
  to active site until the Lys has been
  carbamylated

26

• Key Players
• 3) KaUreG (22D)
• clear sequence similar to nucleotide
  triphosphate-binding proteins, featuring a P-loop
  motif suggests maybe energy dependent step
  during in vivo urease assembly
• 4) KaUreE (18D)
• Metal ion carrier and donor metallochaperones to
  deliver Ni2 ions to KaUreDFG-apo-urease
• The operon also includes the ureA, ureB, and ureC
  genes, encoding the three structural subunits of
  the a3ß3?3 apoenzyme.

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KaUre(ABC)3

• K. aerogenes urease with UreA depicted in blue,
  UreB in orange, and UreC in yellow, together
  forming a (UreABC)3 structure.

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Model for Urease Activation by accessory genes
29
Urease Activation

• In vivo activation of the apoprotein requires the
  presence of nickel ion, carbon dioxide, and
  numerous urease accessory gene products.
• Carbon dioxide molecule that reacts with
  apoprotein to become a nickel ligand.
• Upon activation, UreD, UreE, UreF, and UreG
  dissociate from the enzyme and are recycled

30
Crystal Structure of UreE from B. pasteurii and
K. aerogenes

• Crystal structure of UreE from two different
  species, B. pasteurii and K. aerogenes reveal a
  largely conserved and unique tertiary structure
  made up of two distinct domains, separated by a
  short flexible linker.
• In both species, N-terminal is composed of two
  three-stranded mixed parallel and anti-parallel
  B-sheets stacked upon each other in a nearly
  perpendicular fashion with a short helical region
  between the two sheets.
• C-terminal domain is organized in a ßaßßaß -fold
  and hydrophobic.

31
BpUreE
32
KaUreE
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Metal-ion Coordination

• Zn2 or Ni2 ion in BpUreE through coordination
  of two His100Ne ,
• while a Cu2 is bound to the homologous His96 in
  KaUreE
• KaUreE has a second metal-binding site
  constituted by His110 and His112, resulting in
  two additional Cu2 ions however, this second
  binding site is absent in BpUreE because His110
  and His112 is substituted by Tyr and Lys,
  suggesting that this binding site is not
  functionally relevant.

34
C-Terminal Tail

• In BpUreE, the last few residues on C-terminal
  tail of each monomer are not visible because of
  disorder, probably due to dimerization of
  functional dimmer in the solid state.
• In the KaUreE, the region is altered by a
  truncation of the protein sequence, to eliminate
  15 residues, 10 of which are histidines.
• Further studies necessary to determine the role
  of this terminal region in functional binding of
  the metal ion to UreE.

35
Conclusion

• Urease catalyzes the hydrolysis of urea to yield
  ammonia and carbamate, which spontaneously
  decomposes to yield another molecule of ammonia
  and carbonic acid.
• In solution, the released carbonic acid and the
  two molecules of ammonia are in equilibrium with
  their deprotonated and protonated forms. The net
  effect of these reactions is an increase in pH.
• It serves as a virulence factor in human and
  animal infections of the urinary and
  gastrointestinal tracts.

36
Diseases linked to Urease

• Urease activity plays a central role in the
  pathogenesis. This enzyme has been involved in
  urolithiasis (stone formation), pylonephritis,
  ammonia encephalopathy, hepatic encephalopathy,
  and hepatic coma.
• Urolithiasis. Infection-induced stones result
  from urease-mediated urea hydrolysis in urine.
  Generation of ammonia from cleavage of urea,
  present in high concentration (0.4 to 0.5 M) in
  urine, results in an elevation of pH and
  precipitation of normally soluble polyvalent ions
  present in urine.