1 / 21

Structural features that govern enzymatic activity of Carbonic

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 Rizzello Ñ , Michele Maffia Ñ , Luigi Bubacco *

clea
Download Presentation

Structural features that govern enzymatic activity of Carbonic

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. 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 dell’INFN - INFN, c.p. 13, Frascati, Italy Ñ Department of Biology, University of Lecce, Lecce, Italy

  2. 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 )

  3. 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 >> loss / strong decrease of enzymatic activity

  4. 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 30°C

  5. 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

  6. Sequence analysis: High activity cytosolic CA conservation in : Vertebrata:an average of60 % aminoacidic identity Fish: an average of72 %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 activityCA is more similar to CAII (67% vs 60% CAI, 57% CAIII) expecially in RC (89%, vs 80% CAI, 70% CAIII) >> so we consider mammalian CAII as our reference mammalian CA

  7. 15 Å 10 Å ZN Conservation in Mammalia (CAII), Teleostei, Vertebrata 'extended RC' (aa within 10 Å from Zinc) : % id ~ 90 % for vertebrateCA 'XANES RC' (~ 7 Å from Zinc) : % id = 100% for vertebrate CA

  8. Template selection: PDB reference for computations • Metap server: 3D-jury scoring/ranking alghoritm • 1flj CA III S- glutathiolated Rattus Norvegicus R[Å]=1.80Å • 1v9i CA II bos taurus, Q 253>C R[Å]=2.5 Å • 2cba CA II Homo sapiens (CA2h) R[Å]=1.54Å • 12ca CA2h Ala 121> Val 121R[Å]=2.40 Å • 1hcb CAI Homo sapiens, with bicarbonateR[Å]=1.54Å 2cba (CAII) vs 1flj (CAIII): > 2cba is best resolved (1.54 A) > CAiceRC : more similar to mammalianCAIIthanCAIII >> Choice of 2cba

  9. XANES experimental data A = Icefish CA (CAice) B = Human CA (CA2h)

  10. We generate hundred of theoretical spectra by moving atomic coordinates (The potential is calculated at each step ) Minimization of error function By comparison with exp. data we can fit relevant structural parameters THE MXAN METHOD : • Initial geometrical configurations (2cba) • Exp. data (Xanes spectra)

  11. 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): >> structural parameters with more impact 1) HOH263-Zn: distanceandThetaangle 2) Thr199:Theta angle (Oδ) and distance 3) Coordinated His: distance

  12. Final fitting for CA2h and CAice Bestfit CA2h (Х2 = 4,04) Bestfit CAice (Х2 = 4,44)

  13. Final structural data CAhuman: atomsmore closer to Zinc (average - 0.05Å) 1) HOH263: significantly closer to Zinc in CA2h 2) Oγ (Thr199) : closer in CA2h, consistently with the closer HOH263

  14. HOH263-Zinc distance: effect on the fit Human CA (A) ; Icefish CA (B)

  15. Structure of the reaction centers Thr199 Blue = CAice Red = CA2h Zn2+ HOH263 Coordinated water: 1)CAice ( > Zn-OH distance): >> higher pKa >> lower nuclephilicity pH-bond network:2)CAice (HOH263 and Thr199 closer and shifted consistently) >> first H-bonding position more distant to the Metal

  16. Validation: SAVS ( www.doe-mbi.ucla.edu/Servicies) Protein report (MOE) Score finale (Errat): 95,600 (100 max teorico) Homology modeling Template for modelling : 2cba Modelling with SwissModel/DeepView3.7 and MOE N-term: 1) 2cba has lower resolution (higher uncertainty on first 30positions) 2) Lower conservation between 2cba and CAice

  17. Surface Electrostatic Potential distribution V(S) CAice CA2h entrance to the enzymatic cleft Extimated values (Hex4.5) : in vacuo assumption CA2h: V=+0.62 mVCAice: V= -0.23 mV >> Icefish: 1) negative potential 2) high number of net-charged residues in surface proximity

  18. Surface Electrostatic Potential distribution V(S) CA2h CAice >> Icefish: negative potential around the entrance to the enzymatic cleft

  19. Surface potential distribution V(S) : Icefish peculiarity? Onchorinchusmykiss C.hamatus Icefish (-0,23 mV) Onchorinchus (+0,60 mV) Tribolodon (+0,34 mV) Zebrafish (+0,49 mV) >> Negative V(S): Icefish peculiarity Net formal charge -7 (+3 average for CA fish) icefish Tribolodonhakonensis Daniorerio

  20. 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 >> 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: >> Control on the enzymatic activity: selective pressure on extra-RC positions for chemical-physical properties distribution?

  21. Aknowledgements: We thank Dr. I. Ascone for the excellent support at the LURE facility

More Related