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• Structural features that govern enzymatic
  activity of Carbonic
• anhydrase in a low temperature adapted fish
• Chionodraco hamatus
•  
• Stefano Marino , Kuniko Hayakawa , Keisuke
  Hatada, Maurizio Benfatto, Antonia RizzelloÑ,
• Michele MaffiaÑ, Luigi Bubacco
•  
• Department of Biology, University of Padova,
  Padova, Italy
• Laboratori Nazionali di Frascati dellINFN -
  INFN, c.p. 13, Frascati, Italy
• Ñ Department of Biology, University of Lecce,
  Lecce, Italy

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Carbonic Anhydrase (CA)
Roles of Zinc - Electrostatic catalist
stabilize the negative charged transition from
CO2 to HCO3 - Lower pKa of the coordinated
water ( pH 7 coordinated OH- is an excellent
nucleophile )
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Structure of the reaction center (RC)
Primary ligands His 94, 96,119 HOH
Thr199 H-bond with HOH Glu106 H-bond with
Thr199
Mutations in these RC positions gtgt loss /
strong decrease of enzymatic activity
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Chionodraco hamatus (Icefish) Carbonic Anhydrase
C.hamatus Antartic fish lacking Haemoglobin
and Red blood cells
Activity in f(T) (Maffia,2002) C.hamatus,T.
bernacchii, A. anguilla C.hamatus 1) loss
of activity for T higher than 30C
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Aims of present study on C. hamatus CA (CAice)
1) structure of Reaction Centre (RC)
in Icefish, compared with Human
carbonic anhydrase II (CA2h) as a reference
structure 2) 3D structure of CA
icefish
Tools 1) XAS spectroscopy at
the k-edge of the RC XANES spectra 2)
Molecular Modelling
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Sequence analysis
High activity cytosolic CA conservation in
Vertebrata an average of 60
aminoacidic identity Fish an average of 72
of aacidic identity Mammalian CAII an
average of 74 of aacidic identity
NB a) Mammalia 3 cytosolic isoforms (CAI,
CAII,CAIII) higher activity isoform is CAII
b) Fish high activity CA is more
similar to CAII (67 vs 60 CAI, 57 CAIII)
expecially in RC (89, vs 80 CAI, 70
CAIII) gtgt so we consider mammalian CAII as
our reference mammalian CA
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Conservation in Mammalia (CAII), Teleostei,
Vertebrata
'extended RC' (aa within 10 Å from Zinc) id
90 for vertebrate CA 'XANES RC' ( 7 Å
from Zinc) id 100 for vertebrate CA
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Template selection PDB reference for computations

• Metap server 3D-jury scoring/ranking alghoritm
• 1flj CA III S- glutathiolated Rattus Norvegicus
  RÅ1.80Å
• 1v9i CA II bos taurus, Q 253gtC RÅ2.5 Å
• 2cba CA II Homo sapiens (CA2h) RÅ1.54Å
• 12ca CA2h Ala 121gt Val 121 RÅ2.40 Å
• 1hcb CAI Homo sapiens, with bicarbonate
  RÅ1.54Å

2cba (CAII) vs 1flj (CAIII) gt 2cba is best
resolved (1.54 A) gt CAice RC more
similar to mammalian CAII than CAIII gtgt Choice
of 2cba
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XANES experimental data
A Icefish CA (CAice) B Human CA (CA2h)
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THE MXAN METHOD

• Initial geometrical configurations (2cba)
• Exp. data (Xanes spectra)

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Structural parameter optimization Structural
simulation 64 atoms of the reaction center ( 7
Å from Zinc ) (Zinc, 8HOH,Glu106,Thr199,Thr200,
3His leganti, Phe95, Val143, Glu117)

• gtgt structural parameters with more impact
  
• 1) HOH263-Zn distance and Theta angle
• 2) Thr199 Theta angle (Od) and distance
• 3) Coordinated His distance

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Final fitting for CA2h and CAice
Bestfit CA2h (?2 4,04)
Bestfit CAice (?2 4,44)
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Final structural data
CAhuman atoms more closer to Zinc (average -
0.05 Å)
1) HOH263 significantly closer to Zinc in
CA2h 2) O?
(Thr199) closer in CA2h, consistently with the
closer HOH263
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HOH263-Zinc distance effect on the fit
Human CA (A) Icefish CA (B)
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Structure of the reaction centers
Thr199
Blue CAice Red CA2h
Zn2
HOH263
Coordinated water 1) CAice ( gt Zn-OH
distance) gtgt higher pKa
gtgt lower nuclephilicity
pH-bond network 2) CAice (HOH263 and
Thr199 closer and shifted consistently)
gtgt first H-bonding position more
distant to the Metal
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Homology modeling
Template for modelling 2cba Modelling
with SwissModel/DeepView3.7 and MOE
N-term 1) 2cba has lower resolution
(higher uncertainty on first 30 positions) 2)
Lower conservation between 2cba and CAice
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Surface Electrostatic Potential distribution V(S)
CAice
CA2h
entrance to the enzymatic cleft
Extimated values (Hex4.5) in vacuo
assumption CA2h V 0.62 mV CAice V
-0.23 mV gtgt Icefish 1) negative potential
2) high number of net-charged
residues in surface proximity
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Surface Electrostatic Potential distribution V(S)
CA2h
CAice
gtgt Icefish negative potential around the
entrance to the enzymatic cleft

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1) 100 conservation RC 2) Non conservative
mutations beyond 15 A from Zinc

Sequence analysis
Temperature adaptation ?
Structural effects on the active site
Surface electrostatic potential
different kinetic parameters between CAice and
CA2h
Negative Potential Icefish peculiarity
gtgt control on CA enzymatic activity selective
pressure on extra-RC positions (i.e.
Icefish for precise chemical-physical
properties distribution?)
Considering the 100 aacidic identity in
RC gtgt Control on the enzymatic activity
selective pressure on extra-RC positions for
chemical-physical properties distribution?
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Aknowledgements We thank Dr. I. Ascone for the
excellent support at the LURE facility