Phospholipases d

Phospholipases D

Yani ARHAB – Doctorant Organisation et Dynamique des Membranes Biologiques

Institut de Chimie et Biochimie Moléculaires et Supramoléculaires Bâtiment J. Raulin 1er étage, Domaine scientifique de la Doua 43,

Boulevard du 11 Novembre 1918 69622 VILLEURBANNE Cedex

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Choline

Transphosphatidylation

Phosphatidylalcool

Phosphatidylcholine

R-OH

H-OH

Phosphatidate

Hydrolyse

Activité phospholipase D

2

D

H H D

D

H H D

D

H

H D

D

H H D

Choline

PLD

PC

Intermédiaire acyl-enzyme

Motif N-terminal Motif C-terminal

HO-X

Acide phosphatidique ou

phosphatidylalcool

PLD HKD-Catalysis

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Structure des PLD

PLD de Streptomyces sp. (54 kDa) (Leiros et al., 2000), 1F0I

Partie flexible

Domaine C2

Core

PLD de Brassica oleracea (chou) (90 kDa) modèle SAXS (Stumpe et al., 2007)

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Structure des PLD

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PLD

Choline Oxydase

H₂O₂

O₂

Peroxydase

AAP

HDCBS

λ =510nm

choline Phosphatidate

H₂O

Ca2+

Test Choline Oxydase

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MAQHLLHGTLHATIYEVDALHGGGVMQGFLGKILANVEETIGVGKGETRLYATIDLQKARVGRTRKIKNEP

KNPKWYESFHIYCAHLASDIIFTVKDDNPIGATLIGRAYIPVDQVINGEEVDQWVEILDNDRNPIQGGSKI

HVKLQYFHVEEDRNWNMGIKSAKFPGVPYTFFSQRQGCKVSLYQDAHIPDNFVPRIPLAGGKNYEPQRC

WEDIFDTISNAKHLIYITGWSVYAEIALVRDSRRPKPGGDVTIGELLKKKASEGVRVLLLVWDDRTS

VDVLKKDGLMATHDEETENFFRGSDVHCILCPRNPDDGGSIVQSSQISTMFTHHQKIVVVDSEMP

SRGGSEMRRIVSFVGGIDLCDGRYDTPFHSLFRTLDTVHHDDFHQPNFTGAAITKGGPRE

PWHDIHSRLEGPIAWDVMYNFEQRWSKQGGKDILVKLRDLSDIIITPSPVMFQEDHDVWNV

QLFRSIDGGAAAGFPESPEAAAEAGLVSGKDNITDRSIQDAYIHAIRRAKDFIYVENQ

YFLGSSFAWAADGITPEDINALHLIPKELSLKIVSKIEKGEKFRVYVVVPMWPEGLPGSGSAQAI

LDWQRRTMEMMYKDVIQALRAQGLEEDPRNYLTFFCLGNREVKKDGEYEPAEKPDPDTDYMRAQE

ARRFMIYVHTKMMIVDDEYIIIGSANINQRSMDGARDSEIAMGGYQPHHL

SHRQPARGQIHGFRMSLWYEHLGMLDETFLDPSSLECIEKVNRISDKYWDFYSSESLEHDLPGHLLR

YPIGVASEGDITELPGFEFFPDTKARILGTKSDYLPPILTT

Domaines conservés chez les PLDα végétales

Arabidopsis thaliana PLD α

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Séquences amino-terminales de PLD végétales

At : Arabidopsis thaliana Rc : Ricinus communis (ricin) Gm : Glycine max (soja)

Vu : Vigna unguiculata (cowpea) Bo : Brassica oleracea (chou)

N C C2 HKD HKD

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réfractomètre

Diffusiomètre

Size Exclusion Chromatography

Principe SEC/MALS

Mesure de dn et dc

Mesure de I(θ)

2

scattered

dc

dnMcI

Fractions collectées

Traitement informatique

Séparation des

molécules selon leur

taille

Courbes MALS non traitées

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Références - Abousalham, Rivière, Teissere & Verger 1993, Improved Purification and Biochemical Characterization of Phospholipase D from Cabbage - Abousalham, Teissere, Gardies, Verger & Noat 1995, Phospholipase D from Soybean (Glycine max L.) Suspension-Cultured cells : Purification, Structural and Enzymatic Properties - El Maarouf, Carrière, Rivière & Abousalham 2000, Functional Expression in Insect cells, One-step Purification and Characterization of a Recombinant Phospholipase D from Cowpea (Vigna unguiculata L. Walp) - Imamura & Horiuti 1977, Enzymatic Determination of Phospholipase D Activity with Choline Oxidase - Leiros, Secundo, Zambonelli, Servi & Hough 2000, The First Crystal Structure of a Phospholipase D - Selvy, Lavieri, Lindsley & Brown 2011, Phospholipase D : Enzymology, Functionality and Chemical Modulation - Stumpe, Konig & Ulbrich-Hofmann 2007, Insights into the Structure of Plant α-Type Phospholipase D - Wang, Dyer & Zheng 1993, Purification and Immunological Analysis of Phospholipase D from Castor Bean Endosperm