Title: 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: 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. 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. Two important neurotransmitters involved in the regulation of brain neuronal activity are γ-aminobutyric acid (GABA), 1The abbreviations used are: GABA, γ-aminobutyric acid; GABA-AT, γ-aminobutyric acid aminotransferase; OAT, ornithine aminotransferase; DGD, dialkylglycine decarboxylase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; AAT, aspartate aminotransferase; GEG, γ-ethynyl-GABA, 4-amino-5-hexynoic acid; r.m.s.d., root mean square deviation. 1The abbreviations used are: GABA, γ-aminobutyric acid; GABA-AT, γ-aminobutyric acid aminotransferase; OAT, ornithine aminotransferase; DGD, dialkylglycine decarboxylase; PLP, pyridoxal 5′-phosphate; PMP, pyridoxamine 5′-phosphate; AAT, aspartate aminotransferase; GEG, γ-ethynyl-GABA, 4-amino-5-hexynoic acid; r.m.s.d., root mean square deviation. one of the most widely distributed inhibitory neurotransmitters, and l-glutamic acid, an excitatory neurotransmitter (1.McGeer E.G. McGeer P.L. Thompson S. Hertz L. Kvamme E. McGeer E.G. Schousboe A. Glutamine, Glutamate, and GABA in the Central Nervous System. Liss, New York1983: 3-17Google Scholar). The concentration of GABA is regulated by two pyridoxal 5′-phosphate (PLP)-dependent enzymes, l-glutamic acid decarboxylase, which catalyzes the conversion of l-glutamate to GABA, and GABA aminotransferase (GABA-AT, EC 2.6.1.19), which degrades GABA to succinic semialdehyde (2.Baxter C.F. Roberts E. J. Biol. Chem. 1958; 233: 1135-1139Abstract Full Text PDF PubMed Google Scholar). GABA-AT is a homodimer with each subunit containing an active-site PLP covalently bound to Lys-329 via a Schiff base. The primary sequence of GABA-AT has been deduced from the cDNA of pig brain (3.Kwon O.S. Park J. Churchich J.E. J. Biol. Chem. 1992; 267: 7215-7216Abstract Full Text PDF PubMed Google Scholar) and from peptide fragments of the pig liver enzyme (4.De Biase D. Maras B. Bossa F. Barra D. John R.A. Eur. J. Biochem. 1992; 208: 351-357Crossref PubMed Scopus (20) Google Scholar). Together with the structurally well characterized enzymes ornithine aminotransferase (OAT) and dialkylglycine decarboxylase (DGD), GABA-AT belongs to the subfamily II of the α-family of PLP-dependent enzymes (5.Mehta P.K. Christen P. Adv. Enzymol. Relat. Areas Mol. Biol. 2000; 74: 129-184PubMed Google Scholar). In 1999, the x-ray crystal structure of pig liver GABA-AT was reported at 3.0-Å resolution (6.Storici P. Capitani G. De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (79) Google Scholar), which elucidated the active-site geometry and allowed conclusions to be drawn with respect to its specificity. The mechanism for GABA-AT is well known (7.Cooper A.J.L. Methods Enzymol. 1985; 113: 80-82Crossref PubMed Scopus (22) Google Scholar). GABA is transaminated to succinic semialdehyde, thereby converting the pyridoxal 5′-phosphate (PLP) cofactor to its pyridoxamine 5′-phosphate (PMP) form. In the reverse half-reaction the enzyme is regenerated by conversion of α-ketoglutarate to glutamate.When the concentration of GABA diminishes below a threshold level in the brain, convulsions result (8.Karlsson A. Fonnum F. Malthe-Sorenssen D. Storm-Mathisen J. Biochem. Pharmacol. 1974; 23: 3053-3061Crossref PubMed Scopus (163) Google Scholar), raising the brain GABA levels terminates the seizure (9.Gale K. Epilepsia. 1989; 30: S1-S11Crossref PubMed Scopus (117) Google Scholar). The incidence of seizure activity is very prevalent in the world. In fact, when epilepsy is defined broadly as any disease characterized by recurring convulsive seizures, then almost 1% of the entire world population can be classified as having epilepsy (10.Rogawski M.A. Porter R.J. Pharmacol. Rev. 1990; 42: 223-286PubMed Google Scholar). Consequently, anticonvulsant agents have been sought for centuries. Not until diphenylhydantoin (Dilantin) was introduced onto the drug market over 65 years ago was any particular anticonvulsant drug widely used (11.McNamara J.O. Hardman J.G. Limbird L.E. Molinoff P.B. Ruddon R.W. Gilman A.G. The Pharmacological Basis of Therapeutics. McGraw-Hill, New York1996: 461-486Google Scholar). However, this drug is not generally applicable. In fact, about one-quarter of epileptic patients worldwide (about 12 million people) do not respond to any marketed anticonvulsant drug. Therefore, the need for new anticonvulsant drugs is great (10.Rogawski M.A. Porter R.J. Pharmacol. Rev. 1990; 42: 223-286PubMed Google Scholar).A reduction in the concentrations of GABA has been implicated not only in the symptoms associated with epilepsy (12.Bakay R.A.E. Harris A.B. Brain Res. 1981; 206: 387-404Crossref PubMed Scopus (87) Google Scholar, 13.Loyd K.G. Munati C. Bossi L. Stoeffels C. Talairach J. Morselli P.L. Morselli P.L. Loescher W. Loyd K.G. Neurotransmission, Seizures, Epilepsy. Raven Press, New York1981: 325-338Google Scholar) but also with several other neurological diseases such as Huntington's chorea (14.Butterworth J. Yates C.M. Simpson J. J. Neurochem. 1983; 41: 440-447Crossref PubMed Scopus (34) Google Scholar), Parkinson's disease (15.Nishino N. Fujiwara H. Noguchi-Kuno S.-A. Tanaka C. Jpn. J. Pharmacol. 1988; 48: 331-339Crossref PubMed Scopus (52) Google Scholar), Alzheimer's disease (16.Aoyagi T. Wada T. Nagai M. Kojima F. Harada S. Takeuchi T. Takahashi H. Hirokawa K. Tsumita T. Chem. Pharm. Bull. (Tokyo). 1990; 38: 1748-1749Crossref PubMed Scopus (54) Google Scholar), and tardive dyskinesia (17.Gunne L.M. Haeggstroem J.E. Sjoequist B. Nature (Lond.). 1984; 309: 347-349Crossref PubMed Scopus (216) Google Scholar). Administration of GABA peripherally is not effective, because GABA, under normal conditions, cannot cross the blood-brain barrier; however, several other approaches have been taken to increase the brain concentrations of GABA. One approach is the use of a compound that crosses the blood-brain barrier and then selectively inhibits or inactivates GABA-AT, thereby causing a buildup of GABA. Numerous competitive inhibitors of GABA-AT, particularly compounds having a backbone structure similar to GABA (18.Johnston G.A.R. Curtis D.R. Beart P.M. Game C.J.A. McColloch R.M. Twichin B. J. Neurochem. 1975; 24: 157-160Crossref PubMed Scopus (167) Google Scholar, 19.Schon F. Kelly J.S. Brain Res. 1974; 66: 289-300Crossref Scopus (202) Google Scholar, 20.Johnston G.A.R. Stephanson A.L. Twichin B. J. Pharm. Pharmacol. 1977; 29: 240-241Crossref PubMed Scopus (18) Google Scholar, 21.Brehm L. Hjeds H. Krogsgaard-Larsen P. Acta Chem. Scand. 1972; 26: 1298-1299Crossref PubMed Google Scholar, 22.Krogsgaard-Larsen P. Johnston G.A.R. Lodge D. Curtis D.R. Nature. 1977; 268: 53-55Crossref PubMed Scopus (295) Google Scholar, 23.Beart P.M. Curtis D.R. Johnston G.A.R. Nat. New Biol. 1971; 234: 80-81Crossref PubMed Scopus (52) Google Scholar), and a variety of mechanism-based inactivators (24.Silverman R.B. Methods Enzymol. 1995; 249: 240-283Crossref PubMed Scopus (318) Google Scholar) of GABA-AT (25.Nanavati S.M. Silverman R.B. J. Med. Chem. 1989; 32: 2413-2421Crossref PubMed Scopus (77) Google Scholar) show anticonvulsant activity. The earliest analog reported was 4-amino-5-hexynoic acid (1, γ-ethynyl GABA, GEG), which did not become a clinical candidate (25.Nanavati S.M. Silverman R.B. J. Med. Chem. 1989; 32: 2413-2421Crossref PubMed Scopus (77) Google Scholar). The corresponding alkene, 4-amino-5-hexenoic acid (2, γ-vinyl GABA, vigabatrin) (26.Lippert B. Metcalf B.W. Jung M.J. Casara P. Eur. J. Biochem. 1977; 74: 441-445Crossref PubMed Scopus (356) Google Scholar), was the most effective of these mechanism-based inactivators as an anticonvulsant agent.Vigabatrin (2), which has high potency (27.Loscher W. Neuropharmacology. 1982; 21: 803-810Crossref PubMed Scopus (83) Google Scholar), has been shown to be an effective treatment for epilepsies that are resistant to other anticonvulsant drugs (28.Gidal B.E. Privitera M.D. Sheth R.D. Gilman J.T. Ann. Pharmacother. 1999; 33: 1277-1286Crossref PubMed Scopus (32) Google Scholar) and currently is used in over 60 countries worldwide (but not in the U. S., where it is presently at the pre-registration status for treatment of infantile spasms). However, about 0.5 g of this drug has to be taken daily to be efficacious. As with all psychotropic drugs, there are a variety of side effects, most severe in this case is a visual field defect (29.Kalviainen R. Nousiainen I. CNS Drugs. 2001; 15: 217-230Crossref PubMed Scopus (102) Google Scholar), which restricts the monotherapy of vigabatrin to refractory epilepsy. The drug is sold as a racemic mixture, and it is not known how many of the side effects arise from the administration of the inactive enantiomer (R-isomer). Other mechanism-based inactivators of GABA-AT, such as gabaculine (30.John R.A. Jones E.D. Fowler L.J. Biochem. J. 1979; 177: 721-728Crossref PubMed Scopus (25) Google Scholar, 31.Rando R.R. Bangerter F.W. Biochem. Biophys. Res. Commun. 1977; 76: 1276-1281Crossref PubMed Scopus (73) Google Scholar) and ethanolamine-O-sulfate (32.Fowler L.J. John R.A. Biochem. J. 1972; 130: 569-573Crossref PubMed Scopus (148) Google Scholar), are generally toxic, nonspecific, and/or cross the blood-brain barrier poorly.Recently, it was found that vigabatrin possesses another remarkable activity: it prevents addiction to various abusive substances in rats and baboons (33.Dewey S.L. Morgan A.E. Ashby Jr., C.R. Horan B. Kushner S.A. Logan J. Volkow N.D. Fowler J.S. Gardner E.L. Brodie J.D. Synapse. 1998; 30: 119-129Crossref PubMed Scopus (187) Google Scholar). The mechanism was determined to be the same as that for epilepsy, namely increasing the GABA concentration in the brain by inactivating GABAAT. Increased GABA levels antagonize the extracellular dopamine levels responsible for drug addiction. New inhibitors that are in particular more potent and lipophilic, that is so they can more easily cross the blood-brain barrier, are needed.Two mechanisms were proposed for inactivation of GABA-AT by GEG (34.Burke J.R. Silverman R.B. J. Am. Chem. Soc. 1991; 113: 9329-9340Crossref Scopus (32) Google Scholar); these are shown in Schemes 1 and 2. For inactivation by vigabatrin, three different potential mechanisms (shown in Schemes 3 and 4) were suggested, based on spectroscopic and radiolabeling evidence (35.De Biase D. Barra D. Bossa F. Pucci P. John R.A. J. Biol. Chem. 1991; 266: 20056-20061Abstract Full Text PDF PubMed Google Scholar), chemical intuition (26.Lippert B. Metcalf B.W. Jung M.J. Casara P. Eur. J. Biochem. 1977; 74: 441-445Crossref PubMed Scopus (356) Google Scholar, 36.Metcalf B.W. Biochem. Pharmacol. 1979; 28: 1705-1712Crossref PubMed Scopus (160) Google Scholar), and extensive radiochemical experiments (37.Nanavati S.M. Silverman R.B. J. Am. Chem. Soc. 1991; 113: 9341-9349Crossref Scopus (105) Google Scholar). The controversy regarding the inactivation mechanisms and structures of the covalent adducts could be settled with crystal structures of these inactivators bound to GABA-AT.Here, we have been able to use crystallography and polarized absorption microspectrophotometry to differentiate the inactivation mechanisms and adduct structures for the two mechanism-based inactivators of GABA-AT, GEG (1), and vigabatrin (2). Moreover, we report the presence of a [2Fe-2S] cluster at the center of the dimer, observed in the complex structures as well as in the native enzyme at 2.3-Å resolution.MATERIALS AND METHODSCrystallograph—Pig liver GABA-AT was purified as described previously (35.De Biase D. Barra D. Bossa F. Pucci P. John R.A. J. Biol. Chem. 1991; 266: 20056-20061Abstract Full Text PDF PubMed Google Scholar). Crystals of native enzyme were grown at room temperature in sitting drops by vapor diffusion and microseeding. The protein solution (10-14 mg/ml in 40 mm sodium acetate, pH 5.4) was mixed in a 1:1 ratio with precipitant solution containing 16-18% polyethylene glycol4000, 0-10% glycerol, and 50 mm sodium cacodylate, pH 6.0, and 1 mm dithiothreitol.The GEG-inactivated GABA-AT crystals were obtained by overnight soaking of native crystals in a solution containing 10 mm GEG. Vigabatrin-inactivated GABA-AT crystals were obtained by pre-treatment of native GABA-AT with 10 mm vigabatrin for 1 h prior to crystallization. Each data set was obtained from a single flash-cooled crystal using either 10% ethylene glycol or Panjelly™ (Jena Bioscience) as cryoprotectants. Only a few cycles of crystal annealing were performed to improve crystal mosaicity. Data were collected on MAR image plates, processed with MOSFLM, and scaled with SCALA from the CCP4 suite (38.Collaborative computational project number 4Acta Crystallog. Sect. D. 1994; 50: 760-763Crossref PubMed Scopus (19707) Google Scholar). The models were refined with REFMAC5, including TLS refinement (39.Winn M.D. Isupov M.N. Murshudov G.N. Acta Crystallogr. D. Biol. Crystallogr. 2001; 57: 122-133Crossref PubMed Scopus (1648) Google Scholar). Strong non-crystallographic symmetry restraints were imposed on the positional and thermal parameters of the four monomers in the asymmetric unit. In the initial rounds of refinement, the cofactor and Lys-329 were set as dummy values. This was followed by cyclic averaging using DM (40.Cowtan K. Joint CCP4 and ESF-EACBM Newsletter on Protein Crystallography. 1994; 31: 34-38Google Scholar) to obtain the best unbiased map for interpretation of the adducts. After model building, refinement of the final model was performed without torsional angle restraints on the linkages between Lys-329, cofactor, and the inhibitor moiety. Planarity was imposed on the atoms of the pyridine ring, the carboxylate group, and, in case of the GEG complex, on the atoms belonging to the planar system of the CA=C double bond.Chemical Analysis of Iron and Sulfur Content—The amount of iron bound to GABA-AT was determined colorimetrically, by using the iron chelator ferrozine (41.Stookey L.L. Anal. Chem. 1970; 42: 779-781Crossref Scopus (3536) Google Scholar). GABA-AT (5-10 μm, 460 μl) was mixed with ultrapure concentrated HCl (100 μl) and was incubated at 80 °C for 20 min with periodic vortexing. Following centrifugation at 15,000 rpm for 5 min to pellet the denatured protein, the supernatant (510 μl) was mixed with 10 mm ferrozine (20 μl) and 75 mm ascorbic acid (20 μl). To allow ferrozine chelation, the mixture was neutralized with saturated ammonium acetate and incubated for 20 min at room temperature. The absorbance at 562 nm of the newly formed magenta Fe(ligand)32+ species was determined, and the iron concentration was calculated using the extinction coefficient for ferrozine, ϵ = 27,900 m-1 cm-1 (41.Stookey L.L. Anal. Chem. 1970; 42: 779-781Crossref Scopus (3536) Google Scholar). Acid-labile inorganic sulfide was determined by the method of King and Morris (42.King T.E. Morris R.O. Methods Enzymol. 1967; 10: 631-641Google Scholar).Crystal Microspectrometry—Crystals of GABA-AT were stored at 15 °C in a stabilizing solution containing 22% (w/v) polyethylene glycol 4000, 50 mm sodium citrate, and 1 mm dithiothreitol at pH 5.6. The GABA-AT complexes were produced by soaking single crystals in the stabilizing solution containing 100 mm GEG or 10 mm vigabatrin, respectively. Crystals were loaded in a quartz flow cell that was mounted on the thermostated stage of a Zeiss MPM03 microspectrophotometer, equipped with a ×10 UV-visible Ultrafluar objective. Single crystal absorption spectra were collected in the range 300-700 nm with the electric vector of the linearly polarized light parallel and perpendicular to the extinction directions, as previously described (43.Mozzarelli A. Peracchi A. Rossi G.L. Ahmed S.A. Miles E.W. J. Biol. Chem. 1989; 264: 15774-15780Abstract Full Text PDF PubMed Google Scholar, 44.Mozzarelli A. Rossi G.L. Annu. Rev. Biophys. Biomol. Struct. 1996; 25: 343-365Crossref PubMed Scopus (104) Google Scholar). The isotropic crystal spectrum Ai of GABA-AT was calculated according to Ai = 1/3(Ax + Ay + Az), where Ax, Ay, and Az are the spectra recorded along the three perpendicular extinction directions (45.Hofrichter J. Eaton W.A. Annu. Rev. Biophys. Bioeng. 1976; 5: 511-560Crossref PubMed Scopus (93) Google Scholar).Coordinates—The coordinates for native GABA-AT, and its complex structures with GEG and vigabatrin, have been deposited in the Rutgers Protein Data Bank (accession codes 1ohv, 1ohy, and 1ohw, respectively).RESULTSOverall Structure—The quality of the native GABA-AT data (2.3 Å) has been considerably improved since the previous study (6.Storici P. Capitani G. De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (79) Google Scholar) due to better crystal order, use of flash-cooled crystals, and data acquisition at a synchrotron radiation source. Crystallographic details are given in Table I. The new structure confirms the previous model. The asymmetric unit contains two physiological dimers (AB and CD) with C2 symmetry. The two dimers superimpose with an r.m.s.d. of 0.23 Å, and the four subunits show pairwise r.m.s.d. values between 0.18 and 0.30 Å. This indicates that, within the limits of error, there is no structural asymmetry between the four different copies in the crystallographic asymmetric unit. Also, considering the good quality of the overall crystallographic parameters (Table I), it appears unlikely that asymmetric dimers have been incorporated stochastically into the crystal lattice. Refinement of global temperature factors (TLS refinement), however, revealed that AB is the more mobile dimer within the crystal lattice. After TLS refinement, the remaining (intrinsic) B-factors of the subunits were virtually identical. This allowed the imposition of strong NCS constraints not only on the positional parameters but also on the individual B-factors.Table IData collection and refinement statisticsData setNativeVigabatrin complexGEG complexSampleNative GABA-ATGABA-AT co-crystallized with 10 mm vigabatrinGABA-AT soaked in 10 mm GEG for 24 hX-ray source; λ(Å)ESRF, Grenoble; 0.984Elettra, Trieste; 0.984Rotating anode; 1.542Unit cell: a, b, c (Å)68.6, 225.0, 70.369.7, 226.7, 71.469.0, 225.9, 70.3β (o)108.4108.8108.5Resolution range (Å)30.0—2.330.0—2.330.0—2.8Number of reflections80,29788,76742,802Rsym (%)aRsym = ΣhklΣi(|I(hkl) — 〈I(hkl)〉|)/ΣhklΣi 〈I(hkl)〉8.57.39.8〈I〉/σ(I)5.96.76.6Completeness (%)90.096.185.7Multiplicity2.02.22.5Rfactor (%)bRfactor is the conventional R factor18.819.419.8Rfree (%)cRfree is the R factor calculated with 5% of the data that were not used for refinement22.122.422.9Number of protein atoms29,21629,21629,216Number of cofactor atoms152424Number of FeS atoms888Number of water molecules641739337Mean overall B-factor (Å2)dIn parentheses, residual overall B-factor after TLS refinement24.0 (18.1)26.3 (11.9)25.9 (9.2)r.m.s.d. bond lengths (Å)er.m.s.d. from ideal stereochemistry0.0090.0100.012r.m.s.d. bond angles (o)er.m.s.d. from ideal stereochemistry1.11.21.4a Rsym = ΣhklΣi(|I(hkl) — 〈I(hkl)〉|)/ΣhklΣi 〈I(hkl)〉b Rfactor is the conventional R factorc Rfree is the R factor calculated with 5% of the data that were not used for refinementd In parentheses, residual overall B-factor after TLS refinemente r.m.s.d. from ideal stereochemistry Open table in a new tab Scheme 1View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 2View Large Image Figure ViewerDownload Hi-res image Download (PPT)The Native Cofactor: Structure and Absorption Spectrum— All four copies of the PLP cofactor in the asymmetric unit form a Schiff base linkage with Lys-329 (Fig. 1) and exhibit similar B-factors (between 15 and 20 Å2) after TLS refinement. With a torsional angle κ (for torsion angle definition and atom nomenclature see Fig. 4C) of 23° ± 3° the Schiff's base nitrogen is roughly in the plane of the pyridine ring and forms a short H-bond with the pyridine oxygen (O3) indicative of a protonated aldimine. The torsional angle around the formal C4′=N double bond is 140° ± 2°. Thus, the conformation appears to be rather strained.Fig. 1Stereographic projection of the active site of GABA-AT. The final model is shown together with the 2Fo - Fc map (contour level, 1.2 σ). An acetate molecule is found close to Arg-192, i.e. at a position where the carboxylate moiety of the natural substrate GABA is expected to bind (6.Storici P. Capitani G. De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (79) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4A, stereographic projection of the active site of GABA-AT after chemical modification with GEG. The final model is shown together with the cyclic averaged omit maps (contour level, 1.0 σ). The ternary adduct has been modeled according to structure 10 of Scheme 2. B, stereographic projection of the active site of GABA-AT after chemical modification with vigabatrin. The final model is shown together with the cyclic averaged omit maps (contour level, 1.0 σ). The ternary adduct has been modeled according to structure 15 of Scheme 3. C, comparison of adduct structures. The models of the native PLP cofactor forming an aldimine linkage with Lys-329 (carbon atoms colored in cyan) of the ternary adduct in the GABA-AT·GEG complex (carbons in gray), and of the ternary adduct in the GABA-AT·vigabatrin complex (carbons in magenta) are shown. For this comparison the active-site protein residues have been superimposed. Side chains Phe-189 and Arg-192 are found slightly shifted upon formation of the ternary complexes. The schematic drawing defines the torsional angle κ and the nomenclature of the atoms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Single crystal polarized absorption spectra of GABA-AT, recorded along three perpendicular directions (Fig. 2), exhibit an intense band centered at 412 nm. This band is usually attributed to the ketoenamine tautomer of the protonated internal aldimine (i.e. with the proton on the aldimine nitrogen). However, there is a second band centered between 333 and 338 nm. A band at a similar position has been found in several PLP-dependent enzymes (43.Mozzarelli A. Peracchi A. Rossi G.L. Ahmed S.A. Miles E.W. J. Biol. Chem. 1989; 264: 15774-15780Abstract Full Text PDF PubMed Google Scholar, 46.Phillips R.S. Demidkina T.V. Zakomirdina L.N. Bruno S. Ronda L. Mozzarelli A. J. Biol. Chem. 2002; 277: 21592-21597Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and has been attributed to the enolimine tautomer of the internal aldimine (with the proton on the pyridine oxygen), a species stabilized by a more hydrophobic environment (47.Faeder E.J. Hammes G.G. Biochemistry. 1970; 9: 4043-4049Crossref PubMed Scopus (80) Google Scholar). Noteworthy, the position of the minor band changes with the polarization direction, which suggests that this band does not originate from a single electronic transition, but results from overlapping bands. The reconstructed isotropic spectrum of the crystal (Fig. 2, inset) is similar to the spectrum of the enzyme in solution (35.De Biase D. Barra D. Bossa F. Pucci P. John R.A. J. Biol. Chem. 1991; 266: 20056-20061Abstract Full Text PDF PubMed Google Scholar) with respect to both the peak positions and relative intensities, indicating that crystallization did not perturb the coenzyme environment or the tautomeric equilibrium.Fig. 2Polarized absorption spectra of native GABA-AT crystals. Spectra were recorded with the polarization direction along the three mutually perpendicular extinction directions of the monoclinic GABA-AT crystals and are shown with distinct line-types. Inset, calculated isotropic spectrum.View Large Image Figure ViewerDownload Hi-res image Download (PPT)[2Fe-2S] Cluster—Unexpectedly, a [2Fe-2S] cluster was identified on the molecular 2-fold symmetry axis at the center of the dimer in all structures investigated (Fig. 3, A and B). Previously, it was not possible to resolve this feature because of poor resolution (3 Å) (6.Storici P. Capitani G. De Biase D. Moser M. John R.A. Jansonius J.N. Schirmer T. Biochemistry. 1999; 38: 8628-8634Crossref PubMed Scopus (79) Google Scholar). Fig. 3B shows that cysteines 135 and 138 at the N-terminal end of helix 5 together with their symmetry related mates chelate the cluster. Each of the iron atoms is coordinated tetrahedrally by two cysteines of one subunit and the two free sulfur atoms, which lie on the molecular 2-fold axis and cross-link to the symmetry related second iron atom. The Fe-S distances are 2.2-2.3 Å. The B-factors of the cluster atoms and the cysteines match well (differences < 4 Å2).Fig. 3The [2Fe-2S] cluster at the center of the GABA-AT dimer.A, structure of the GABA-AT dimer. The view is approximately along the molecular 2-fold symmetry axis. The Cα traces of the two subunits are shown in black and red. Helix 5 and its symmetry mate are highlighted by thick gray traces. The [2Fe-2S] cluster on the molecular 2-fold symmetry axis together with the liganding cysteines and the two symmetry related PLP cofactors are shown in full view. B, close-up view of A. The Cα traces have been omitted for clarity. Symmetry-related residues are marked with the symbol "#." C, stereographic close-up view. The molecular 2-fold axis is approximately along the vertical direction. The native 2Fo - Fc omit map (magenta; contour level 1.2 σ) and the anomalous difference map (light blue; contour level 4.5 σ) were computed with data to 2.3-Å resolution (data set of the vigabatrin complex). The iron and sulfur atoms were not included for phasing.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 3View Large Image Figure ViewerDownload Hi-res image Download (PPT)Scheme 4View Large Image Figure ViewerDownload Hi-res imag