Structures of γ-Aminobutyric Acid (GABA) Aminotransferase, a Pyridoxal 5′-Phosphate, and [2Fe-2S] Cluster-containing Enzyme, Complexed with γ-Ethynyl-GABA and with the Antiepilepsy Drug Vigabatrin

Abstract

␥-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, ␥-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-Å resolution) and in complex with vigabatrin as well as with the close analogue ␥-ethynyl-GABA (to 2.3 and 2.8 Å, 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 radiolabeling 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.

Cellular and subcellular localizations of nonheme ferric and ferrous iron in the rat brain: a light and electron microscopic study by the perfusion-Perls and -Turnbull methods

“…Similarly, the deep cerebellar nuclei receive GABAergic projections from Purkinje cells. Recent studies revealed that GABA transaminase has an iron-sulfur cluster at the center of the GABA transaminase dimer (Storici et al, 2004). This enzyme is synthesized in postsynaptic neurons and astrocytes (reviewed in Kugler, 1993) and plays an essential role in the degradation of GABA, which is released from GABAergic synaptic terminals.…”

Enantiomers of 4-Amino-3-fluorobutanoic Acid as Substrates for γ-Aminobutyric Acid Aminotransferase. Conformational Probes for GABA Binding

“…FlexX performs flexible docking using an incremental fragment construction method to place a flexible ligand into a rigid protein structure. The receptor structure used was the X-ray crystal structure of two homodimers of pig liver GABA-AT in complex with vigabatrin (4-vinyl-GABA) (PDB code: 1OHW) (44). Water molecules were removed as they were found not to be located within the active site region.…”

Structural Determinants of the β-Selectivity of a Bacterial Aminotransferase

“…Reflections were indexed and integrated using XDS (13), and scaling and merging of the data were done with the program SCALA (14) from the CCP4 Software Suite (15). Phaser (16) was used for molecular replacement with a mixed input model generated by the FFAS03 server (17) on the basis of the structures of glutamate-1-semialdehyde 2,1-aminomutase from Thermus thermophilus strain HB8 (Protein Data Bank code 2E7U), D-phenylglycine aminotransferase from Pseudomonas stutzeri strain ST-201 (code 2CY8), and 4-aminobutyrate aminotransferase from pig (code 1OHV (18)). The resulting model was subjected to successive rounds of automatic model building with ARP/wARP (19), followed by manual model building in Coot (20).…”