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

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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