Structures of gamma-aminobutyric acid (GABA) aminotransferase, a pyridoxal 5'-phosphate, and [2Fe-2S] cluster-containing enzyme, complexed with gamma-ethynyl-GABA and with the antiepilepsy drug vigabatrin.
Gamma-aminobutyric acid aminotransferase (GABA-AT) is a pyridoxal 5'-phosphate-dependent enzyme responsible for the degradation of the inhibitory neurotransmitter GABA. GABA-AT is a validated target for antiepilepsy drugs because its selective inhibition raises GABA concentrations in brain. The antiepilepsy drug, gamma-vinyl-GABA (vigabatrin) has been investigated in the past by various biochemical methods and resulted in several proposals for its mechanisms of inactivation. In this study we solved and compared the crystal structures of pig liver GABA-AT in its native form (to 2.3-A resolution) and in complex with vigabatrin as well as with the close analogue gamma-ethynyl-GABA (to 2.3 and 2.8 A, respectively). Both inactivators form a covalent ternary adduct with the active site Lys-329 and the pyridoxal 5'-phosphate (PLP) cofactor. The crystal structures provide direct support for specific inactivation mechanisms proposed earlier on the basis of radio-labeling experiments. The reactivity of GABA-AT crystals with the two GABA analogues was also investigated by polarized absorption microspectrophotometry. The spectral data are discussed in relation to the proposed mechanism. Intriguingly, all three structures revealed a [2Fe-2S] cluster of yet unknown function at the center of the dimeric molecule in the vicinity of the PLP cofactors.
Storici
Paola
P
Division of Structural Biology, Biozentrum, University of Basel, Switzerland.
De Biase
Daniela
D
Bossa
Francesco
F
Bruno
Stefano
S
Mozzarelli
Andrea
A
Peneff
Caroline
C
Silverman
Richard B
RB
Schirmer
Tilman
T
D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000378:metabolism*; D000927:Anticonvulsants / Q000737:chemistry*; D018360:Crystallography, X-Ray; D007091:Image Processing, Computer-Assisted; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D011732:Pyridoxal Phosphate / Q000737:chemistry / Q000378:metabolism*; D020888:Vigabatrin / Q000737:chemistry*; D005680:gamma-Aminobutyric Acid / Q000031:analogs & derivatives / Q000737:chemistry*
D000927:Anticonvulsants; D007506:Iron-Sulfur Proteins; C482076:gamma-ethynyl-aminobutyric acid; D005680:gamma-Aminobutyric Acid; D011732:Pyridoxal Phosphate; D000612:4-Aminobutyrate Transaminase; D020888:Vigabatrin