Structural features that govern enzymatic activity of Carbonic anhydrase in a low temperature adapted fish Chionodraco hamatus Stefano Marino *, Kuniko.

Presentation on theme: "Structural features that govern enzymatic activity of Carbonic anhydrase in a low temperature adapted fish Chionodraco hamatus Stefano Marino *, Kuniko."— 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 Rizzello   Michele Maffia , Luigi Bubacco * * Department of Biology, University of Padova, Padova, Italy + Laboratori Nazionali di Frascati dell’INFN - INFN, c.p. 13, Frascati, Italy  Department of Biology, University of Lecce, Lecce, Italy

2 Roles of Zinc: - Electrostatic catalist CO 2 to HCO 3 ¯ - Electrostatic catalist: stabilize the negative charged transition from CO 2 to HCO 3 ¯ - Lower pKa( pH 7: coordinated OH - excellent nucleophile ) - Lower pKa of the coordinated water ( pH 7: coordinated OH - is an excellent nucleophile ) Carbonic Anhydrase (CA):

3 Structure of the reaction center (RC): Primary ligands: His 94, 96,119 ; HOH Thr199 Thr199: H-bond with HOH Glu106 Glu106: H-bond with Thr199 Mutations in these RC positions >> loss / strong decrease of enzymatic activity

4 Activity in f(T) (Maffia,2002): C.hamatus,T. bernacchii, A. anguillaC.hamatus: 1) loss of activity for T higher than 30°C T higher than 30°C Chionodraco hamatus (Icefish) Carbonic Anhydrase: C.hamatus: C.hamatus: Antartic fish lacking Haemoglobin and Red blood cells

5 Aims of present study on C. hamatus CA (CAice): 1) structure of Reaction Centre (RC) in Icefish 1) structure of Reaction Centre (RC) in Icefish, compared with Human carbonic anhydrase II (CA2h) as a reference structure 2) 3D structure 2) 3D structure of CA icefish Tools : 1)XANES spectra 1) XAS spectroscopy at the k-edge of the RC : XANES spectra 2) Molecular Modelling

6 Sequence analysis: High activity cytosolic CA conservation in : High activity cytosolic CA conservation in : Vertebrata: an average of 60 % aminoacidic identity Fish72 % Fish: an average of 72 % of aacidic identity Mammalian CAII: 74 % Mammalian CAII: an average of 74 % of aacidic identity NB: a) Mammalia: NB: a) Mammalia: 3 cytosolic isoforms (CAI, CAII,CAIII); higher activity isoform is CAII Fish 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) >> so we consider mammalian CAII as our reference mammalian CA >> so we consider mammalian CAII as our reference mammalian CA

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

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 121 R[Å]=2.40 Å 1hcb CAI Homo sapiens, with bicarbonate R[Å]=1.54Å 2cba (CAII) vs 1flj (CAIII): > 2cba is best resolved (1.54 A) > 2cba is best resolved (1.54 A) > CAice RC : more similar to mammalian CAII than CAIII > CAice RC : more similar to mammalian CAII than CAIII >> Choice of 2cba

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

10 THE MXAN METHOD : We generate hundred of theoretical spectra by moving atomic coordinates (The potential is calculated at each step ) By comparison with exp. data we can fit relevant structural parameters Minimization of error function 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: distance and Theta angle Thr199 2) Thr199: Theta angle (Oδ) and distance 3) Coordinated His: distance

12 Final fitting for CA2h and CAice CA2h Bestfit CA2h (Х 2 = 4,04) CAice Bestfit CAice (Х 2 = 4,44)

13 Final structural data 1) HOH263: significantly closer to Zinc in CA2h 2) Oγ (Thr199) : closer in CA2h, consistently with the closer HOH263 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 Coordinated water1)CAice( > Zn-OH distance):>> higher pKa Coordinated water: 1) CAice( > Zn-OH distance):>> higher pKa >> lower nuclephilicity pH-bond network:2) pH-bond network: 2) CAice (HOH263 and Thr199 closer and shifted consistently) >> first H-bonding position more distant to the Metal Blue = CAice Red = CA2h Zn 2+ Thr199 HOH263

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

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

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

19 Surface potential distribution V(S) : Icefish peculiarity? icefish Onchorinchus mykiss Tribolodon hakonensis Danio rerio 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)

20 Considering the 100% aacidic identity in RC: >> Control on the enzymatic activity: selective pressure on extra-RC positions for chemical-physical properties distribution? Temperature adaptation ? Structural effects on the active site Surface electrostatic potential different kinetic parameters between CAice and CA2h Negative Potential: Icefish peculiarity Sequence analysis + 1) ~100 % conservation RC 2) Non conservative mutations: beyond 15 A from Zinc beyond 15 A from Zinc >> control on CA enzymatic activity: selective pressure on extra-RC positions (i.e. Icefish : for precise chemical-physical properties distribution?)

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