Structural snapshots of the catalytic cycle of the phosphodiesterase Autotaxin.
Autotaxin (ATX) is a secreted phosphodiesterase that produces the signalling lipid lysophosphatidic acid (LPA). The bimetallic active site of ATX is structurally related to the alkaline phosphatase superfamily. Here, we present a new crystal structure of ATX in complex with orthovanadate (ATX-VO5), which binds the Oγ nucleophile of Thr209 and adopts a trigonal bipyramidal conformation, following the nucleophile attack onto the substrate. We have now a portfolio of ATX structures we discuss as intermediates of the catalytic mechanism: the new ATX-VO5 structure; a unique structure where the nucleophile Thr209 is phosphorylated (ATX-pThr). Comparing these to a complex with the LPA product (ATX-LPA) and with a complex with a phosphate ion (ATX-PO4), that represent the Michaelis complex of the reaction, we observe movements of Thr209, changes in the relative displacement of the zinc ions, and a water molecule that likely fulfils the second nucleophilic attack. We propose that ATX follows the associative two-step in-line displacement mechanism.
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Electronic address: jens.hausmann@mpi-muenster.mpg.de.
Jens
J
Hausmann
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Willem-Jan
WJ
Keune
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Agnes L
AL
Hipgrave Ederveen
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Leonie
L
van Zeijl
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Robbie P
RP
Joosten
Division of Biochemistry, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands. Electronic address: a.perrakis@nki.nl.
Anastassis
A
Perrakis
D000469:Alkaline Phosphatase / Q000737:chemistry; D002384:Catalysis; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006801:Humans; D008055:Lipids / Q000737:chemistry; D008246:Lysophospholipids / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000737:chemistry*; D010766:Phosphorylation; D011487:Protein Conformation; D014638:Vanadates / Q000737:chemistry*; D015032:Zinc / Q000737:chemistry; D000099189:Lysophospholipase D
D000469:Alkaline Phosphatase; D008055:Lipids; D008246:Lysophospholipids; D010710:Phosphates; D010727:Phosphoric Diester Hydrolases; D014638:Vanadates; D015032:Zinc; D000099189:Lysophospholipase D; C032881:lysophosphatidic acid
Alkaline phosphatase; Autotaxin; Catalytic mechanism; Phosphodiesterase; X-ray crystallography