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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
1
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
3
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)
4
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
7
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
8
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
9
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