Ectotherms : Adaptation of enzyme to temperature
Designed and Performed by : Nicolas PUIGMAL, Master 1 BIMoPoDD : Integrated Biology : Molecules, Population
and Sustanaible Development
UNIVERSITE de PERPIGNAN Faculté des Sciences Exactes et Expérimentales
Ectotherms : Adaptation of enzymes to temperature
2
Summary
-Introduction : a review of existing knowledge
-Study on LDH-A in Sphyraena
-Conclusion
UPVDUniversité de Perpignan Via Domitia
Ectotherms : Adaptation of enzymes to temperature
3
Introduction : a review of existing knowledgeUPVD
Université de Perpignan Via Domitia
Ectotherms
Temperature
variationIon transport = ATP
Protection and tissue reparation
(HSP)
Poor insulation
Low energy for reproductionFailure of evolution??
http://www.arkive.org/
Ectotherms : Adaptation of enzymes to temperature
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UPVDUniversité de Perpignan Via Domitia
Failure of evolution?? No, absolutely not …
Adaptation to their environment
Ectothermic metabolic in-between hot and cold
Protein concentration?
Environmental modification? (pH)
Efficient proteins?
??
?
Introduction : a review of existing knowledge
Adaptation to their environment by efficient protein
Ectotherms : Adaptation of enzymes to temperature 5
UPVDUniversité de Perpignan Via Domitia
Study of enzyme kinetics Arrherius
Km
Kcat
George N. Somero, Comparative Biochemistry and Physiology, Part B 139 (2004) 321-333 (modified)
Introduction : a review of existing knowledge
Ectotherms : Adaptation of enzymes to temperature
6
UPVDUniversité de Perpignan Via Domitia
LDH-A : Lactate Deshydrogenase-A-many data on the function, structure, sequence-orthologs in many species : same substrate (pyruvate) and cofactor (NADH)
Preserved active site
StabilityFunctionality
Flexibility « door hinge » zone
Flexibility « door hinge » zone
- Determined by the nature of the amino acids of the hinge
- Role of pH, osmolyte
Introduction : a review of existing knowledge
Ectotherms : Adaptation of enzymes to temperature
7
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Study of the evolution of LDH-A in 6 species of Barracuda (genus Shyraena)Sphyraena argentea (Temperate north)
S. lucasana (Subtropical)
S. idiastes (Temperate south)
S. ensis
S. acutipinnis
S. barracuda
http://geographie.ens.fr/
Ectotherms : Adaptation of enzymes to temperature 8
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Experiments of LDH-A
Extraction from white skeletal muscle : rich in enzyme
Preparation of LDH-A by reductor and alkaline solution
Native LDH-A
Native LDH-A
Modification pH
Modification pH
Denatured LDH-A
Denatured LDH-A
Dithiothreitol (break disulfide
bridges)
Dithiothreitol (break disulfide
bridges)
Denatured LDH-A
Denatured LDH-A
TrypsineTrypsine
Digested LDH-A
Digested LDH-A
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Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Resultats
Sphyraena argentea
S. lucasana
S. idiastes
S. helleri
S. ensis
S. barracuda
-Similar sequence between these three species
-Differences due to adaptation
-Further studies with these three species
Linda Z. Holland and all, Biochemistry,
Vol.36, No.11, 1997 (modified)
Ectotherms : Adaptation of enzymes to temperature
Ectotherms : Adaptation of enzymes to temperature 10
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Manipulation of LDH-A
Synthesis of cDNA
Guanidium Thiucyanate acid : protein
(ex: RNAase)
: Dynabead oligo dT
: Reverse transcriptase
All ARN
: cDNA
: RNAm
: RNA
: DNA
Ectotherms : Adaptation of enzymes to temperature 11
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
PCR amplification
and sequencing
Deduced amino acid
sequences of LDH-A
Resultats
8
61 68
223-1 difference between S.l and S.i: 8th position on the outside of the active site
-S.a: 4 differences : 8,61,68 and 223
Linda Z. Holland and all, Biochemistry, Vol.36, No.11, 1997 (modified)
Ectotherms : Adaptation of enzymes to temperature 12
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Which role have these position? Site Directed Mutagenesis
Km S.a native Km S.l native
Km S.l native = Km S.l Cloned
Km C-8 Km S.a native
Position 8 has no role in the functionality
Km C-61-68 = Km S.a native
Position 61 and 68 have roles in the functionality
Linda Z. Holland and all, Biochemistry, Vol.36, No.11, 1997 (modified)
Ectotherms : Adaptation of enzymes to temperature
13
Study of LDH-A in barracudaUPVD
Université de Perpignan Via Domitia
Which role have these position? Site Directed Mutagenesis
Stability of the protein
C-8 (clone of S.l with same amino acid in position 8 that S.a)
Same stability that S.a
Position 8 play a role in the stability
Increased stability
Linda Z. Holland and all, Biochemistry, Vol.36, No.11, 1997 (modified)
Ectotherms : Adaptation of enzymes to temperature
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ConclusionUPVD
Université de Perpignan Via Domitia
6 species of fishes in differents environment
Adaptative modification of LDH-A
Position 8 Position 61-68 Position 223
Role in the thermal stability
Role in the functionality (kinetic
property)
3 related species
No role…
Peptide Mapping
Site Directed Mutagenesis
Independent
Important role of changes outside the active site for adapatation temperature
Protein sequence analysis
Ectotherms : Adaptation of enzymes to temperature
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UPVDUniversité de Perpignan Via Domitia
Thank you for your attention …
I have my own adaptation to
cold