Crystal structures of Aedes aegypti kynurenine aminotransferase Qian Han1,*, Yi Gui Gao1,2,*, Howard Robinson3, Haizhen Ding1, Scott Wilson2 and Jianyong Li1 1 Department of Pathobiology, University of Illinois, Urbana, IL, USA 2 School of Chemical Sciences, University of Illinois, Urbana, IL, USA 3 Biology Department, Brookhaven National Laboratory, Upton, NY, USA
Keywords aminotransferase; crystal structure; kynurenic acid; kynurenine aminotransferase; mosquito Correspondence J. Li, Department of Pathobiology, University of Illinois, 2001 South Lincoln Avenue, Urbana, IL 61802, USA Fax: +217 2447421 Tel: +217 244–3913 E-mail:
[email protected] *Qian Han and Yi Gui Gao contributed equally to this work. Note The atomic coordinates and structure factors (PDB codes 1YIZ and 1YIY) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ, USA (http://www.rcsb.org).
Aedes aegypti kynurenine aminotransferase (AeKAT) catalyzes the irreversible transamination of kynurenine to kynurenic acid, the natural antagonist of NMDA and 7-nicotinic acetycholine receptors. Here, we report the crystal structure of AeKAT in its PMP and PLP forms at 1.90 and 1.55 A˚, respectively. The structure was solved by a combination of single-wavelength anomalous dispersion and molecular replacement approaches. The initial search model in the molecular replacement method was built with the result of single-wavelength anomalous dispersion data from the BrAeKAT crystal in combination with homology modeling. The solved structure shows that the enzyme is a homodimer, and that the two subunits are stabilized by a number of hydrogen bonds, salts bridges, and hydrophobic interactions. Each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to the prototypical fold type, aminotransferase subgroup I. The three-dimensional structure shows a strictly conserved ‘PLP-phosphate binding cup’ featuring PLPdependent enzymes. The interaction between Cys284 (A) and Cys284 (B) is unique in AeKAT, which might explain the cysteine effect of AeKAT activity. Further mutation experiments of this residue are needed to eventually understand the mechanism of the enzyme modulation by cysteine.
(Received 2 February 2005, accepted 7 March 2005) doi:10.1111/j.1742-4658.2005.04643.x
Aedes aegypti kynurenine aminotransferase (AeKAT) is a multi-function aminotransferase [1]. Its mammalian homolog, KAT-I can catalyze several amino acids and many biologically relevant keto acids [2,3], and is identical to glutamine transaminase K and also a cysteine S-conjugate b-lyase [4–8]. KAT-I is present in the brain [9,10], and also in the kidney and liver [11,12], which indicates the important role
of KAT-I in the bioactivation of environmental pollutants that contribute to liver- and kidney-associated carcinogenesis [2]. Although the kidney and liver show much greater KAT activity than the brain, the emphasis of KAT research has been almost exclusively on enzymes in the CNS, paralleling the investigation into the pivitol role of kynurenic acid (KYNA) therein.
Abbreviations AeKAT, Aedes aegypti kynurenine aminotransferase; KAT, kynurenine aminotransferase; KYNA, kynurenic acid; MAD, multiwavelength anomalous dispersion; PLP, pyridoxal 5-phosphate; PMP, pyridoxamine 5-phosphate; rmsd, root mean square deviation; SAD, single wavelength anomalous dispersion.
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KYNA is a metabolite in the tryptophan metabolic pathway. In mammals, it is synthesized by irreversible transamination of kynurenine by KATs. KYNA represents the only known endogenous antagonist of the excitatory action of ionotropic excitatory amino acids, showing the highest affinities for the glycine modulatory site of the NMDA subtype of glutamate receptor [13–15] and the a7-nicotinic acetylcholine receptor [16– 18]. In mammals, it protects the CNS from overstimulation by excitatory cytotoxins [19,20]. The inhibitory actions of KYNA at excitatory amino acid KAI receptors underlie its neuroprotective [19–22] and anticonvulsant effects [23,24]. Fluctuations in endogenous brain KYNA levels significantly influence neuronal excitation and vulnerability to excitotoxic attack [25–28]. In addition, KYNA is also involved in maintaining physiological arterial blood pressure [29–33]. The role of activity-dependent synaptic plasticity in learning and memory is a central issue in neuroscience. Much of the relevant experimental work concerns the possible role of long-term potentiation in learning. Most forms of long-term potentiation are glutamatergic and the most prominent form is induced following activation of the NMDA receptor [34]. KYNA, as the only natural antagonist of NMDA, may, therefore, be involved in the processes of memory and learning in the CNS. Savvateeva et al. [35] demonstrated that the mutant cardinal fly (3-hydroxykynurenine excess, local KYNA level might be affected) shows a decline in learning and memory, which implies a possible role for KYNA in the formation of long-term potentiation. Direct evidence is, however, still missing. The physiological importance of KYNA has attracted a considerable amount of attention towards understanding the molecular regulation of KYNA production in living organisms. KATs have become the target enzymes when studying modulation of the KYNA level in a number of pathological conditions in animals. A. aegypti KAT (AeKAT) shares 45–50% sequence identity with mammalian or human KAT-Is [36]. Functional characterization of its recombinant protein, expressed in a baculovirus ⁄ insect cell-expression system, showed that the protein is active to kynurenine [36]. The protein showed high activity towards many biologically relevant keto acids. Interestingly, most keto acids showed substrate inhibition at relatively high concentrations. Cysteine had an intriguing effect on the enzyme activity towards kynurenine, inducing enhancement at relative low concentrations and inhibition at higher concentrations [1]. Moreover, AeKAT is mainly expressed in adult heads, indicating its major function in the CNS [36]. A bacterial homolog and human KAT-I have been systematically characterized FEBS Journal 272 (2005) 2198–2206 ª 2005 FEBS
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using their respective recombinant enzymes [3,37], and both three-dimensional structures have recently been solved [38,39]. The biochemical comparison of the three enzymes has been discussed previously [3]. To understand the catalytic mechanism and structural basis underlying these biochemical differences, it is essential that the three-dimensional structure of AeKAT is determined and a comparative study with mammalian KATs is carried out. Here, we provide data that describe the crystal structure of AeKAT as obtained using macromolecular crystallography.
Results and Discussion Crystallization, single wavelength anomalous dispersion modeling, and homology modeling Single wavelength anomalous dispersion (SAD) diffraction data for a Br-AeKAT (PMP form) derivative were collected at cryogenic temperature at X12C at the National Synchrotron Radiation Source in Brookhaven National Laboratory (BNL) (k ¼ 0.97 A˚). The Br-AeKAT (PMP form) crystal has an orthorhombic unit cell with parameters of a ¼ 55.34 A˚, b ¼ 95.32 A˚, c ¼ 167.67 A˚, and diffracts to 1.90 A˚ resolution. The space group has been determined as P212121 from an auto-index using gadds program. Two molecules of AeKAT are in an asymmetric unit, based on calculation of the Matthews coefficient [40]. SAD diffraction data of the Br-AeKAT (native ⁄ PLP form) derivative were collected in the same manner (k ¼ 1.1 A˚). The PLP form crystal has an orthorhombic unit cell with parameters of a ¼ 55.28 A˚, b ¼ 94.98 A˚, c ¼ 167.60 A˚ and diffracts to 1.55 A˚ resolution (Table 1). An initial atomic model with 520 residues of two molecules was obtained based on the SAD data of BrAeKAT crystals with a resolution at 1.90 A˚ (Fig. 1A). Because the SAD model has only 60% residues assigned, we turned to a molecular replacement method using homology modeling. The first model was built by homology modeling using the initial SAD model and two homology structures (PDB codes: 1gck and 1v2d) as template structures. Briefly, SAD coordinates were assigned to target residues of AeKAT, the structures of other residues were built based on two search models. Loop areas were highly optimized to target protein AeKAT using the program insight ii (Accelrys). In total 100 models were built, 10 of which were used in the initial refinement tests based on the procheck [41] results. Only one model (Fig. 1B) was used in further refinement using shelx-97, o and x-plor. This strategy enabled us to solve the three-dimensional structure of AeKAT. Figure 1 shows the initial SAD model (A), a 2199
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Table 1. Crystal parameters, data collection and refinement statistics of AeKAT. Data collection
PLP form
PMP form
Space group Unit cell (a, b, c)
P212121 55.28, 94.98, 167.60 1.55 924 984 128 458 5.7 (29.1) 7.2 (6.7) 99.3 (99.8)
P212121 55.34, 95.32, 167.67 1.90 716,820 68,715 6.3 (19.6) 10.4 (9.8) 97.1 (90.5)
26.13 25.42 25.4 27.9 0.010 2.41 1.88
22.37 21.85 21.8 26.4 0.007 1.72 2.08
1.49
1.65
2 · 3315 2 · 15 5 441 18.43 23.24 20.82 15.57 27.33
2 · 3315 2 · 16 5 440 18.26 23.78 20.99 12.41 26.97
Resolution (A˚) Observation reflections Unique reflections Rmerge (%) Redundancy Completeness (%) Refinement R-all (%) R-observed (%) R-work (%) R-free (%) RMS bond lengths (A˚) RMS bond angles () All-atom RMS fit for the two chains (A˚) Ca-only RMS fit for the two chains (A˚) No. of protein atoms No. of PLP ⁄ PMP atoms No. of heavy atoms (Br-) No. of solvent molecules (water) Average B factor main chain (A˚2) Average B side chain (A˚2) Average B over all (A˚2) Average B factor PLP ⁄ PMP (A˚2) Average B factor solvet (water) (A˚2)
homology model (B) and the final refined structure of the PMP form (C) of AeKAT. Overall structure The PMP form of AeKAT was solved at a resolution of 1.90 A˚. The three-dimensional structure of the AeKAT
PLP form was solved using its PMP structure as an initial refinement model by shelx-97 and x-plor at a resolution of 10 to 1.55 A˚. A homodimer is present in the asymmetric unit. Excellent electron density allowed the modeling of 418 of 429 residues, 2 PLPs and 441 solvent molecules in the PLP form structure. Both forms have the same structure except that there is no bond formed between the cofactor and Lys255 in the PMP form. The stereochemistry of the model was assessed using procheck [41]. In both the PLP and PMP forms, 87% of the residues were in the most favored regions of the Ramachandran plot. Although Tyr286 (A) and (B) in both forms fall within a disallowed region of the Ramachandran plot of the solved structures, the excellent electron density allowed us to unambiguously assign the observed conformation. The protein architecture revealed by AeKAT consists of the prototypical fold of aminotransferases subgroup I [42,43], characterized by an N-terminal arm, and a small and a large domain (Fig. 2). The N-terminal arm consists of a random coiled stretch made up of residues 12–26, the small domain (residues 27–52 and 310–429) folds into a five-stranded parallel and antiparallel b sheet surrounded by four a helices. The large domain (residues 53–309) adopts an a ⁄ b structure that resembles the Rossmann fold, which shows conserved a ⁄ b topology, in which a sharply twisted sevenstranded b-sheet inner core is nested into a conserved array of nine a helices which are contributed by both the interior and external of the molecule. As observed in other subgroup I aminotransferases, the functional unit of AeKAT consists of a homodimer with subunits related by a dyad axis to its two active sites located at the domain interface in each subunit, and at the subunit interface in the dimer (Fig. 2). Many hydrogen bonds are formed between two subunits, i.e. Lys17–Gln119 (2.63, 2.68 A˚), Lys17–Val122 (2.69, 2.89 A˚), Asn71–Trp262 (2.89, 2.95 A˚), Asn71– Gly261 (2.87, 2.95 A˚), Trp72–Thr260 (2.52, 3 A˚),
Fig. 1. Line ribbon representation of initial SAD model, homology model and final refined model. (A) Initial SAD model. (B) Homology model. (C) Final refined model of the PMP form. The molecules are viewed down the molecular twofold axis. The two subunits are colored blue and green, respectively.
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Fig. 2. Stereo ribbon representation of the AeKAT molecule. The small and large domains of one subunit are given in green and blue, respectively. Both N-terminal arms are grey, and other subunit is red. The cleft hosting the enzyme active site can be seen at the domain interface where the PLP cofactor is shown as a ball-and-stick. The C-terminus and N-terminus are indicated as C-ter and N-ter, respectively.
Trp72–Ser258 (2.84, 2.95 A˚), Tyr73–Thr250 (2.87, 3.9 A˚), Tyr111–Gln282 (2.79, 2.95 A˚), two Tyr115 (2.57 A˚), and Gly261–Thr290 (2.78, 2.85 A˚) of the opposite subunits in PLP form. Several hydrophobic interactions participate in the stabilization of the homodimer, in particular, the interactions between Phe46 and Leu69 (3.57, 4.39 A˚) and the two Val108 (4 A˚) of the opposite subunits. Moreover, the N-terminal arms also contribute towards the stability of the AeKAT dimer. Two salt bridges are established between Asp112 and Lys6 (3.96, 4.15 A˚) and Asp112 and Arg7 (3.48, 3.55 A˚) of the opposite subunits. In AeKAT structures, Cys284 (A) and Cys284 (B) with distances of 3.46 A˚ (PLP form) and 3.57 A˚ (PMP form) form a thiol–thiolate hydrogen bond, which is unique in AeKAT (Fig. 5B). The AeKAT active site Similar to human KAT-I, AeKAT possesses two active sites located around the local dyad axis. Each active site contains one PLP molecule and is hosted in a deep cleft at the domain interface made up of residues from both subunits (Fig. 2). Both PLP and PMP can be identified with confidence, with clear electronic density maps of the structures (Fig. 3). Each PLP cofactor sits within a binding pocket defined by two regions contributed by residues from the large domains of both subunits (Fig. 2). The bottom of the PLP-binding pocket is entirely defined by residues from the large domain of the corresponding monomer. With the exception of Lys255 and Gly110, all these residues are at, or close FEBS Journal 272 (2005) 2198–2206 ª 2005 FEBS
Fig. 3. Diagrams of 2Fo – Fc electron density maps for the active sites of the PMP and PLP forms. The map contoured at 2.0 sigma is calculated using data between 10.0 and 1.90 A˚ and 10.0 and 1.55 A˚ resolution for the PMP and PLP forms of AeKAT, respectively. (A) PLP form; (B) PMP form.
to, the edge of the inner core b sheet, pointing toward the domain interface and facing the re-face of the PLP ring. Distinct arrays of residues form the lateral walls 2201
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Fig. 4. Schematic diagram showing active site interactions in AeKAT. Hydrogen bonds are shown by dotted lines. Phe135 and Val223 sandwich the pyridine ring of PLP. (A) PLP form; (B) PMP form.
of the PLP-binding pocket. In particular, the stretch 43–46, together with the side chains of Arg405, Asn189 and Asn193 of the large domain, make up one pocket wall. The opposite wall consists of residues Tyr111 and Tyr138 of the same monomer and Phe286, His287 and Tyr73 of the other subunit. In the PLP form, AeKAT carries the PLP molecule covalently bound in the active site by Schiff base linkage to the catalytic Lys255 (Figs 3B and 4A), resulting in the formation of an internal aldimine bond (C4a ¼ Nz) with an angle of 105.5 at the pyridine ring of PLP. Several other residues contact the PLP molecule and participate in its recognition and binding. The phosphate group of the cofactor is engaged in a number of interactions with residues making up the strictly conserved ‘PLP-phosphate binding cup’, featuring PLP-dependent enzymes [44]. In particular, its OP1 oxygen forms a set of hydrogen bonds with Ser252 (2.97 A˚), Ala110 (with its backbone nitrogen atom at 2.78 A˚) and the solvent molecule W120 at a distance of 2.82 A˚. PLP OP2 forms a hydrogen bond with Tyr73 (B) (in other subunit, 2.55 A˚) and the solvent molecule W20 (2.73 A˚). Finally, the PLP OP3 atom interacts with Lys263 (at 2.73 A˚ from the Nz atom) and with the backbone nitrogen atom of Tyr111 (at 2.87 A˚). The PLP phenolic oxygen is held in place by interactions with Tyr224 (at 2.56 A˚ from its OH atom) and Asn193 (at a distance of 2.62 A˚ with its OD1 2202
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atom). Moreover, the N1 atom of the PLP pyridine moiety forms hydrogen bonds with the carboxylic oxygen atoms of Asp221 (at 2.66 A˚). Several hydrophobic interactions further stabilize PLP. In particular, Phe135 and Val223 surround the anthranilic moiety of the cofactor, on its si- and re-face, respectively (Fig. 4A). In the PMP form, there is no internal aldimine bond between Lys255 and PMP and the distance between PMP amine and Lys255 Nz is 3.45 A˚ (Fig. 3B). The interactions in the active site are similar to the PLP form (Figs 3A and 4B). It is interesting to find a thiol–thiolate hydrogen bond formed between two subunits in AeKAT, which might be a target for understanding AeKAT regulation. Changes in the sulfur oxidation state of cysteine residues influence the activity of many proteins [45,46]. Reversible disulfide bond formation and the associated conformation changes are likely to play an important role in cellular redox regulation. In particular, disulfide bond formation between distant cysteines may be an effective mechanism for the induction of conformational changes that lead to switches in protein activity [47–49]. In human mitochondrial branched chain aminotransferase, the redox-active dithiol ⁄ disulfide Cys315-Xaa-Xaa-Cys318 center has been proposed in the regulation of enzyme activity. Cys315 appears to be the sensor for redox regulation of the enzyme activity, whereas Cys318 acts as the ‘resolving cysteine’, allowing for reversible formation of a disulfide bond [50,51]. AeKAT has a similar sequence, Cys284-XaaXaa-Xaa-Cys288 (Fig. 5A), but there is no thiol–thio-
Fig. 5. Putative cysteine regulation site of AeKAT. (A) Partial alignment result of human KAT-I and AeKAT. (B) Diagrams of 2Fo ) Fc electron density maps for Cys284 (A) and Cys284 (B) and surrounding residues. The map was contoured at 2.0 sigma.
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late hydrogen bond between the two cysteine residues (9.4 A˚) in the AeKAT structure. To understand the mode of enzyme regulation by cysteine, we comparatively analyzed human KAT-I and AeKAT. First, AeKAT activity can be stimulated by cysteine [1], whereas the human homolog is not positively affected by cysteine [3]. Second, sequence alignment showed that AeKAT had two additional cysteine residues compared with human KAT-I, as indicated in Fig. 5A (green background), the equivalent of Cys284 in AeKAT is Ser276 in the human enzyme. Third, there are no thiol–thiolate hydrogen bonds or disulfide bonds in the human KAT-I structure [39], whereas in the AeKAT structure, Cys284 is located in the large domain of the homodimer, the structure shows evidence of a thiol–thiolate hydrogen bond between Cys284 (A) and Cys284 (B) (Fig. 5B), which is close to the active center. Under oxidizing conditions, these cysteine residues in AeKAT can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.46 A˚ in the PLP form and 3.57 A˚ in the PMP form), requiring a decrease of only 1.5–1.6 A˚. Thus, residue Cys284 is most likely the cysteine-regulation target of AeKAT. Further mutation experiments of this residue along with biochemical and structural analyses are needed to eventually understand the mechanism of the enzyme modulation by cysteine.
Experimental procedures Expression and purification of recombinant AeKAT AeKAT lacking the N-terminal mitochondrial leader sequence (amino acids 1–48) was expressed in a baculovirus ⁄ insect cell protein expression system, and purified by DEAE Sepharose, phenyl Sepharose, hydroxyapatite, and native PAGE separation (PMP form) or gel filtration (PLP ⁄ native form) [36]. To begin with, we did not pay attention to the enzyme forms, and obtained only the PMP form of AeKAT, because the running buffer for native PAGE has 192 mm glycine. Later, when we used gel filtration as the last step of purification, we obtained the native form of AeKAT. The proposed reaction for aminotransferases is shown in Scheme 1. The purity of the protein was
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assessed by SDS ⁄ PAGE analysis. Protein concentration was determined by a BioRad protein assay kit using bovine serum albumin as a standard. The purified recombinant AeKAT was concentrated to 10 mgÆmL)1 protein in 5 mm phosphate buffer, pH 7.5 using a Centricon YM-30 concentrator (Millipore, Billerica, MA, USA).
AeKAT crystallization Initial crystallization screening was performed using Hampton Research Crystal Screens (Hampton Research, Laguna Niguel, CA, USA) with sitting-drop and hanging-drop vapor diffusion methods with the volume of reservoir solution at 500 lL and the drop volume at 5 lL, containing 2.5 lL of protein sample and 2.5 lL of reservoir solution. AeKAT crystals were obtained in a solution containing 10 mgÆmL)1 of protein, 30% (w ⁄ v) PEG 1000, and 0.1 m of Tris ⁄ HCl at pH 8.5. Refinement of preliminary crystallization conditions resulted in the growth of quality crystals in a solution containing 5 mgÆmL)1 protein, 15% (w ⁄ v) PEG 1000, and 0.1 m Tris ⁄ HCl at pH 8.5. Single crystals for suitable X-ray analysis appeared in 4 days and grew to maximum sizes of 0.5 · 0.3 · 0.2 mm3 in 3 weeks at 4 C.
Data collection and processing To solve the AeKAT structure through multiwavelength anomalous diffusion (MAD) [52] and single-wavelength anomalous diffusion (SAD) [53], we first tried l-selenomethionine-labeled AeKAT, but failed to obtain quality diffraction data from its crystals. Subsequently, the Br-AeKAT derivative was generated by soaking AeKAT crystals in 1 m NaBr for 5–10 s followed by transferr of the NaBr-AeKAT crystals to a cryoprotectant solution containing mother liquid [1 m NaBr, and 25% (v ⁄ v) glycerin in Tris ⁄ HCl pH 8.5] for 30–60 s [54]. For X-ray analyses, oscillation diffraction images of Br-AeKAT were obtained using a Bruker General Area Detector Diffraction System (Madison, WI, USA) equipped with a fourcircle diffractometer and a HiStar multiwire area detector. Individual AeKAT crystals were frozen using 10% sucrose plus 30% PEG 400 as a cryoprotectant solution in order to prevent the appearance of ice diffraction during data collection at cryogenic temperatures. Diffraction data for Br-AeKAT crystals were collected at the Brookhaven National Synchrotron Light Source X12C beamline (wavelengths k ¼ 0.97 A˚). Single crystals were exposed to a cold nitrogen stream during data collection using a MAR research 165 mm CCD detector. The PEG 400, sucrose, or glycerin provided sufficient cryoprotection during data collection. All data were auto-indexed and integrated using hkl software [55], scaling and merging of diffraction data was performed using scalepack [56]. The parameters of crystal and data collection are listed in Table 1.
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Structure determination Initial phases for the PMP crystal form were obtained from a SAD experiment. The structure of the PMP form was determined by the molecular replacement method with the homology module in insight ii (Accelrys) using the partial SAD model and two search models, Thermus thermophylus aspartate aminotransferase (Protein Data Bank code 1gck) [57] and glutamine aminotransferase (Protein Data Bank code 1v2d) [38] as template structures. The program amore [58] was used to calculate both cross-rotation and translation functions in the 10–3.0 A˚ resolution range. The initial model was subjected to iterative cycles of crystallographic refinement with the programs x-plor [59], shelx-97 [60] and with graphic sessions for model building using the program o [61]. A random sample containing 1000 reflections was set apart to calculate the free R-factor [62]. Solvent molecules were manually added at positions with density > 1.5 sigma in the 2Fo ) Fc map, considering only peaks engaged in at least one hydrogen bond with a protein atom or a solvent atom. The procedure converged to an R-factor and free R-factor of 0.218 and 0.264, respectively, with ideal geometry. Residues of the two subunits in AeKAT are numbered 12 (A) to 429 (A) and 12 (B) to 429 (B), respectively. The results of refinement are summarized in Table 1.
Acknowledgements We thank Dr John M. Sanders, Department of Chemistry, University of Illinois at Urbana-Champaign, for his help in our homology model building using Insight II. This work was supported by Grant AI 44399 from the National Institutes of Health and the work was carried out in part at the National Synchrotron Light Source, Brookhaven National Laboratory.
References 1 Han Q & Li J (2004) Cysteine and keto acids modulate mosquito kynurenine aminotransferase catalyzed kynurenic acid production. FEBS Lett 577, 381– 385. 2 Cooper AJ (2004) The role of glutamine transaminase K (GTK) in sulfur and alpha-keto acid metabolism in the brain, and in the possible bioactivation of neurotoxicants. Neurochem Int 44, 557–577. 3 Han Q, Li J & Li J (2004) pH dependence, substrate specificity and inhibition of human kynurenine aminotransferase I. Eur J Biochem 271, 4804–4814. 4 Perry SJ, Schofield MA, MacFarlane M, Lock EA, King LJ, Gibson GG & Goldfarb PS (1993) Isolation and expression of a cDNA coding for rat kidney cytosolic cysteine conjugate beta-lyase. Mol Pharmacol 43, 660–665.
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5 Mosca M, Cozzi L, Breton J, Speciale C, Okuno E, Schwarcz R & Benatti L (1994) Molecular cloning of rat kynurenine aminotransferase: identity with glutamine transaminase K. FEBS Lett 353, 21–24. 6 Alberati-Giani D, Malherbe P, Kohler C, Lang G, Kiefer V, Lahm HW & Cesura AM (1995) Cloning and characterization of a soluble kynurenine aminotransferase from rat brain: identity with kidney cysteine conjugate beta-lyase. J Neurochem 64, 1448–1455. 7 Schmidt W, Guidetti P, Okuno E & Schwarcz R (1993) Characterization of human brain kynurenine aminotransferases using [3H]kynurenine as a substrate. Neuroscience 55, 177–184. 8 Baran H, Okuno E, Kido R & Schwarcz R (1994) Purification and characterization of kynurenine aminotransferase I from human brain. J Neurochem 62, 730–738. 9 Cooper AJ, Abraham DG, Gelbard AS, Lai JC & Petito CK (1993) High activities of glutamine transaminase K (dichlorovinylcysteine beta-lyase) and omegaamidase in the choroid plexus of rat brain. J Neurochem 61, 1731–1741. 10 Okuno EF, Ishikawa T, Tsujimoto M, Nakamura M, Schwarcz R & Kido R (1990) Purification and characterization of kynurenine-pyruvate aminotransferase from rat kidney and brain. Brain Res 534, 37–44. 11 Cooper AJ, Nieves E, Rosenspire KC, Filc-DeRicco S, Gelbard AS & Brusilow SW (1988) Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine (amide) in rat liver. J Biol Chem 263, 12268– 12273. 12 Cooper AJ & Meister A (1974) Isolation and properties of a new glutamine transaminase from rat kidney. J Biol Chem 249, 2554–2561. 13 Leeson PD & Iversen LL (1994) The glycine site on the NMDA receptor: structure–activity relationships and therapeutic potential. J Med Chem 37, 4053–4067. 14 Perkins MN & Stone TW (1982) An iontophoretic investigation of the actions of convulsant kynurenines and their interaction with the endogenous excitant quinolinic acid. Brain Res 247, 184–187. 15 Birch PJ, Grossman CJ & Hayes AG (1988) Kynurenic acid antagonises responses to NMDA via an action at the strychnine-insensitive glycine receptor. Eur J Pharmacol 154, 85–87. 16 Pereira EF, Hilmas C, Santos MD, Alkondon M, Maelicke A & Albuquerque EX (2002) Unconventional ligands and modulators of nicotinic receptors. J Neurobiol 53, 479–500. 17 Hilmas C, Pereira EF, Alkondon M, Rassoulpour A, Schwarcz R & Albuquerque EX (2001) The brain metabolite kynurenic acid inhibits alpha7 nicotinic receptor activity and increases non-alpha7 nicotinic receptor expression: physiopathological implications. J Neurosci 21, 7463–7473.
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Q. Han et al.
18 Alkondon M, Pereira EF, Yu P, Arruda EZ, Almeida LE, Guidetti P, Fawcett WP, Sapko MT, Randall WR, Schwarcz R et al. (2004) Targeted deletion of the kynurenine aminotransferase II gene reveals a critical role of endogenous kynurenic acid in the regulation of synaptic transmission via alpha7 nicotinic receptors in the hippocampus. J Neurosci 24, 4635–4648. 19 Stone TW (2001) Kynurenic acid antagonists and kynurenine pathway inhibitors. Expert Opin Invest Drugs 10, 633–645. 20 Stone TW (2001) Kynurenines in the CNS: from endogenous obscurity to therapeutic importance. Prog Neurobiol 64, 185–218. 21 Moroni F (1999) Tryptophan metabolism and brain function: focus on kynurenine and other indole metabolites. Eur J Pharmacol 375, 87–100. 22 Schwarcz R (1993) Metabolism and function of brain kynurenines. Biochem Soc Trans 21, 77–82. 23 Foster AC & Fagg GE (1984) Acidic amino acid binding sites in mammalian neuronal membranes: their characteristics and relationship to synaptic receptors. Brain Res 319, 103–164. 24 Scharfman HE, Goodman JH & Schwarcz R (2000) Electrophysiological effects of exogenous and endogenous kynurenic acid in the rat brain: studies in vivo and in vitro. Amino Acids 19, 283–297. 25 Carpenedo R, Chiarugi A, Russi P, Lombardi G, Carla V, Pellicciari R, Mattoli L & Moroni F (1994) Inhibitors of kynurenine hydroxylase and kynureninase increase cerebral formation of kynurenate and have sedative and anticonvulsant activities. Neuroscience 61, 237–243. 26 Cozzi A, Carpenedo R & Moroni F (1999) Kynurenine hydroxylase inhibitors reduce ischemic brain damage: studies with (m-nitrobenzoyl)-alanine (mNBA) and 3,4dimethoxy-[-N-4-(nitrophenyl) thiazol-2yl]-benzenesulfonamide (Ro 61–8048) in models of focal or global brain ischemia. J Cereb Blood Flow Metab 19, 771– 777. 27 Erhardt S, Blennow K, Nordin C, Skogh E, Lindstrom LH & Engberg G (2001) Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurosci Lett 313, 96–98. 28 Battaglia G, Rassoulpour A, Wu HQ, Hodgkins PS, Kiss C, Nicoletti F & Schwarcz R (2000) Some metabotropic glutamate receptor ligands reduce kynurenate synthesis in rats by intracellular inhibition of kynurenine aminotransferase II. J Neurochem 75, 2051–2060. 29 Kiely JM & Gordon FJ (1994) Role of rostral ventrolateral medulla in centrally mediated pressor responses. Am J Physiol 267, H1549–H1556. 30 Ito S, Komatsu K, Tsukamoto K & Sved AF (2000) Excitatory amino acids in the rostral ventrolateral medulla support blood pressure in spontaneously hypertensive rats. Hypertension 35, 413–417.
FEBS Journal 272 (2005) 2198–2206 ª 2005 FEBS
Mosquito kynurenine aminotransferase structure
31 Kapoor V, Kapoor R & Chalmers J (1994) Kynurenic acid, an endogenous glutamate antagonist, in SHR and WKY rats: possible role in central blood pressure regulation. Clin Exp Pharmacol Physiol 21, 891–896. 32 Kapoor V, Thuruthyil SJ & Human B (1998) Reduced kynurenine aminotransferase-I activity in SHR rats may be due to lack of KAT-Ib activity. Neuroreport 9, 1431– 1434. 33 Kwok JB, Kapoor R, Gotoda T, Iwamoto Y, Iizuka Y, Yamada N, Isaacs KE, Kushwaha VV, Church WB, Schofield PR et al. (2002) A missense mutation in kynurenine aminotransferase-1 in spontaneously hypertensive rats. J Biol Chem 277, 35779–35782. 34 Martin SJ, Grimwood PD & Morris RG (2000) Synaptic plasticity and memory: an evaluation of the hypothesis. Annu Rev Neurosci 23, 649–711. 35 Savvateeva E, Popov A, Kamyshev N, Bragina J, Heisenberg M, Senitz D, Kornhuber J & Riederer P (2000) Age-dependent memory loss, synaptic pathology and altered brain plasticity in the Drosophila mutant cardinal accumulating 3-hydroxykynurenine. J Neural Transm 107, 581–601. 36 Fang J, Han Q & Li J (2002) Isolation, characterization, and functional expression of kynurenine aminotransferase cDNA from the yellow fever mosquito, Aedes aegypti (1). Insect Biochem Mol Biol 32, 943–950. 37 Hosono A, Mizuguchi H, Hayashi H, Goto M, Miyahara I, Hirotsu K & Kagamiyama H (2003) Glutamine: phenylpyruvate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8. J Biochem (Tokyo) 134, 843–851. 38 Goto M, Omi R, Miyahara I, Hosono A, Mizuguchi H, Hayashi H, Kagamiyama H & Hirotsu K (2004) Crystal structures of glutamine: phenylpyruvate aminotransferase from Thermus thermophilus HB8: induced fit and substrate recognition. J Biol Chem 279, 16518–16525. 39 Rossi F, Han Q, Li J, Li J & Rizzi M (2004) Crystal structure of human kynurenine aminotransferase I. J Biol Chem 279, 50214–50220. 40 Matthews BW (1968) Solvent content of protein crystals. J Mol Biol 33, 491–497. 41 Laskowski RA, Macarthur MW, Moss DS & Thornton JM (1993) procheck – a program to check the stereochemical quality of protein structures. J Appl Crystallogr 26, 283–291. 42 Jansonius JN (1998) Structure, evolution and action of vitamin B-6-dependent enzymes. Curr Opin Struct Biol 8, 759–769. 43 Nakai T, Okada K, Akutsu S, Miyahara I, Kawaguchi S, Kato R, Kuramitsu S & Hirotsu K (1999) Structure of Thermus thermophilus HB8 aspartate aminotransferase and its complex with maleate. Biochemistry 38, 2413–2424. 44 Denesyuk AI, Denessiouk KA, Korpela T & Johnson MS (2002) Functional attributes of the phosphate group
2205
Mosquito kynurenine aminotransferase structure
45
46
47
48 49
50
51
52
binding cup of pyridoxal phosphate-dependent enzymes. J Mol Biol 316, 155–172. Giles NM, Watts AB, Giles GI, Fry FH, Littlechild JA & Jacob C (2003) Metal and redox modulation of cysteine protein function. Chem Biol 10, 677–693. Xu D, Rovira II & Finkel T (2002) Oxidants painting the cysteine chapel: redox regulation of PTPs. Dev Cell 2, 251–252. Lee C, Lee SM, Mukhopadhyay P, Kim SJ, Lee SC & Ahn WS., YuMH, Storz G & Ryu SE (2004) Redox regulation of OxyR requires specific disulfide bond formation involving a rapid kinetic reaction path. Nat Struct Mol Biol 11, 1179–1185. Georgiou G (2002) How to flip the (redox) switch. Cell 111, 607–610. Choi H, Kim S, Mukhopadhyay P, Cho S, Woo J, Storz G & Ryu S (2001) Structural basis of the redox switch in the OxyR transcription factor. Cell 105, 103–113. Conway ME, Poole LB & Hutson SM (2004) Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme. Biochemistry 43, 7356–7364. Conway ME, Yennawar N, Wallin R, Poole LB & Hutson SM (2002) Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase. Biochemistry 41, 9070–9078. Hendrickson WA, Horton JR & LeMaster DM (1990) Selenomethionyl proteins produced for analysis by multiwavelength anomalous diffraction (MAD): a vehicle for direct determination of three-dimensional structure. EMBO J 9, 1665–1672.
2206
Q. Han et al.
53 Dauter Z, Dauter M & Dodson E (2002) Jolly SAD. Acta Crystallogr D Biol Crystallogr 58, 494–506. 54 Dauter Z, Dauter M & Rajashankar KR (2000) Novel approach to phasing proteins: derivatization by short cryo-soaking with halides. Acta Crystallogr D Biol Crystallogr 56, 232–237. 55 Otwinowski Z & Minor W (1997) Processing of X-ray diffraction data collected in oscillation mode. In Methods in Enzymology (Carter CW & Sweet RM, eds), pp. 307–326, Academic Press, London. 56 Minor W (1993) XDISPLAYF Program. Purdue University, West Lafayette, IN. 57 Ura H, Nakai T, Kawaguchi SI, Miyahara I, Hirotsu K & Kuramitsu S (2001) Substrate recognition mechanism of thermophilic dual-substrate enzyme. J Biochem (Tokyo) 130, 89–98. 58 Navaza J (2001) Implementation of molecular replacement in amore. Acta Crystallogr D Biol Crystallogr 57, 1367–1372. 59 Brunger AT (1992) X-PLOR. Yale University Press, New Haven, CT. 60 Sheldrick GM & Schneider TR (1997) High resolution refinement. Methods Enzymol 277, 319–343. 61 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47, 110–119. 62 Brunger AT (1992) Free R-value – a novel statistical quantity for assessing the accuracy of crystal-structures. Nature 355, 472–475.
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