Paola Storici

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

γ-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, γ-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 γ-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 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.

Structures of GABA aminotransferase, a pyridoxal 5'-phosphate and [2Fe-2S] cluster containing enzyme, complexed with -EthynylGABA and with the antiepilepsy drug vigabatrin

γ-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, γ-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 γ-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 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.

Crystallization and preliminary crystallographic investigations of rhodanese from Azotobacter vinelandii.

The rhdA gene identified in Azotobacter vinelandii codes for a protein, RhdA, which displays rhodanese (thiosulfate-cyanide sulfurtransferase) activity. RhdA was overexpressed and purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2 with unit-cell parameters a = 44.4, b = 150.8, c = 53.8 A; on a synchrotron source the diffraction patterns could be collected to a resolution limit of 1.8 A. Evaluation of the crystal density indicates that the crystal lattice accommodates one molecule per asymmetric unit and that the solvent content is 59% of the total volume.

Engineering methionine γ-lyase from Citrobacter freundii for anticancer activity

Methionine deprivation of cancer cells, which are deficient in methionine biosynthesis, has been envisioned as a therapeutic strategy to reduce cancer cell viability. Methionine γ-lyase (MGL), an enzyme that degrades methionine, has been exploited to selectively remove the amino acid from cancer cell environment. In order to increase MGL catalytic activity, we performed sequence and structure conservation analysis of MGLs from various microorganisms. Whereas most of the residues in the active site and at the dimer interface were found to be conserved, residues located in the C-terminal flexible loop, forming a wall of the active site entry channel, were found to be variable. Therefore, we carried out site-saturation mutagenesis at four independent positions of the C-terminal flexible loop, P357, V358, P360 and A366 of MGL from Citrobacter freundii, generating libraries that were screened for activity. Among the active variants, V358Y exhibits a 1.9-fold increase in the catalytic rate and a 3-fold increase in KM, resulting in a catalytic efficiency similar to wild type MGL. V358Y cytotoxic activity was assessed towards a panel of cancer and nonmalignant cell lines and found to exhibit IC50 lower than the wild type. The comparison of the 3D-structure of V358Y MGL with other MGL available structures indicates that the C-terminal loop is either in an open or closed conformation that does not depend on the amino acid at position 358. Nevertheless, mutations at this position allosterically affects catalysis.

Molecular bases of PDE4D inhibition by GEBR-library compounds for cognitive amelioration in Alzheimer’s disease

Selected members of the large rolipram-related GEBR family of phosphodiesterase-4 (PDE4) inhibitors have been shown to facilitate long term potentiation (LTP) and improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, little if any structure-activity relationship studies have so far been carried out in order to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degr...Selected members of the large rolipram-related GEBR family of type 4 phosphodiesterase (PDE4) inhibitors have been shown to facilitate long-term potentiation and to improve memory functions without causing emetic-like behavior in rodents. Despite their micromolar-range binding affinities and their promising pharmacological and toxicological profiles, few if any structure-activity relationship studies have been performed to elucidate the molecular bases of their action. Here, we report the crystal structure of a number of GEBR library compounds in complex with the catalytic domain of PDE4D as well as their inhibitory profiles for both the long PDE4D3 isoform and the catalytic domain alone. Furthermore, we assessed the stability of the observed ligand conformations in the context of the intact enzyme using molecular dynamics simulations. The longer and more flexible ligands appear to be capable of forming contacts with the regulatory portion of the enzyme, thus possibly allowing some degree of selectivity between the different PDE4 isoforms.

GABA-aminotransferase, a target for antiepileptic drug therapy

gamma-Aminobutyrate aminotransferase (GABA-AT), a pyridoxal phosphate-dependent enzyme, is responsible for the degradation of the inhibitory neurotransmitter GABA and is a target for antiepileptic drugs because its selective inhibition raises GABA concentrations in brain. The X-ray structure of pig GABA-AT has been determined to 3.0 A resolution by molecular replacement with the distantly related enzyme ornithine aminotransferase. Both omega-aminotransferases have the same fold, but exhibit side chain replacements in the closely packed binding site that explain their respective specificities. The aldimines of GABA and the antiepileptic drug vinyl-GABA have been modeled into the active site.