Why study nitrilases

1
Why study nitrilases?

• The glu,cys,lys catalytic triad offers versatile
  chemistry
• Use of enzymes in the manufacture of fine
  chemicals is attractive because of
  enantioselectivity.
• Use of enzymes in environmental remediation is
  attractive because organisms may not survive the
  toxins.
• Their metabolic role is not well characterised

2
Chemistry
Nitrilase - cyanide dihydratase - CynD - B.
pumilus, P.stutzeri
Cyanide hydratase - G. sorghi
Amidase G. pallidus
Carbamylase 1ERZ Agrobacterium sp.
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The Superfamily
4
Relationship of solved structures to the
bacterial and plant nitrilases, CynD, and fungal
cyanide hydratrases
Plant nitrilases
Solved structures
CynD
Bacterial nitrilases
Fungal cyanide hydratases
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Current uses

• Environmental remediation of cyanide
• Manufacture of acrylic acid
• Manufacture of specific enantiomers R-mandelic
  acid, R-4-cyano-3-hydroxybutyric acid,
  S-phenyllactic acid, (S)-ibuprofen,
  (S)-a-phenylglycine and (S)-naproxen.

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Oligomerization

• All known nitrilase homologues are at least
  dimers
• There are four crystal structures of homologues
  two are dimers and two are tetramers with 222
  symmetry. Only one has known enzyme activity a
  carbamylase
• Our published work has shown three forms built
  around helical symmetry two are terminating
  spirals with 14 and 18 subunits respectively. The
  18mer changes to an open helix at pH5.4
• Most determinations of oligomeric state are based
  on gel-filtration and are unreliable but the
  numbers range from 2-18 subunits for all known
  nitrilases

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Link between activity and oligomerization

• Nagasawa et al and two previous authors have
  shown that dimers are inactive until they
  oligomerize (in Rhodococci )
• We have shown that in case of B. pumilus an
  activity increase accompanies the pH dependent
  increase in size
• We have shown that destructive mutation of
  residues in the interfaces which associate to
  form helices abolishes activity
• Mueller et al 2006 archaeal nitrilase active as
  a dimer

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What is it useful to know?(biotechnology
perspective)

• The configuration of the active site
• How is specificity / activity controlled
• The oligomeric configuration
• How is the oligomeric configuration controlled
• How the oligomeric configuration affects the
  active site configuration