The crystal structure of human a -amino- b -carboxymuconate- e -semialdehyde decarboxylase in complex with 1,3-dihydroxyacetonephosphate suggests a regulatory link between NAD synthesis and glycolysis Silvia Garavaglia 1 , Silvia Perozzi 1 , Luca Galeazzi 2 , Nadia Raffaelli 2 and Menico Rizzi 1 1 DiSCAFF Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, University of Piemonte Orientale ‘A. Avogadro’, Novara, Italy 2 Department of Molecular Pathology and Innovative Therapies, Section of Biochemistry, Universita ` Politecnica delle Marche, Ancona, Italy Introduction In humans, tryptophan at a level that exceeds the basal requirements for protein and serotonin synthesis is oxi- datively degraded through the kynurenine pathway, producing the highly unstable intermediate a-amino- b-carboxymuconate-e-semialdehyde (ACMS) [1]. As shown in Fig. 1, ACMS can be either non-enzymati- cally converted into quinolinic acid (QA), fuelling NAD biosynthesis, or transformed by the action of ACMS decarboxylase (ACMSD, also known as picoli- nate carboxylase; EC 4.1.1.45) into a-aminomuconic Keywords cerebral malaria; kynurenine pathway; metal-dependent amidohydrolase; NAD biosynthesis; neurological disorders Correspondence M. Rizzi, DiSCAFF, University of Piemonte Orientale, Via Bovio 6, 28100 Novara, Italy Fax: +39 0321 375821 Tel: +39 0321 375712 E-mail: rizzi@pharm.unipmn.it Database The atomic coordinates and structure factors of hACMSD have been deposited with the Protein Data Bank (http:// www.rcsb.org) with accession codes 2wm1 and r2wm1, respectively (Received 1 July 2009, revised 8 September 2009, accepted 10 September 2009) doi:10.1111/j.1742-4658.2009.07372.x The enzyme a-amino-b-carboxymuconate-e-semialdehyde decarboxylase (ACMSD) is a zinc-dependent amidohydrolase that participates in picolinic acid (PA), quinolinic acid (QA) and NAD homeostasis. Indeed, the enzyme stands at a branch point of the tryptophan to NAD pathway, and deter- mines the final fate of the amino acid, i.e. transformation into PA, com- plete oxidation through the citric acid cycle, or conversion into NAD through QA synthesis. Both PA and QA are key players in a number of physiological and pathological conditions, mainly affecting the central ner- vous system. As their relative concentrations must be tightly controlled, modulation of ACMSD activity appears to be a promising prospect for the treatment of neurological disorders, including cerebral malaria. Here we report the 2.0 A ˚ resolution crystal structure of human ACMSD in complex with the glycolytic intermediate 1,3-dihydroxyacetonephosphate (DHAP), refined to an R-factor of 0.19. DHAP, which we discovered to be a potent enzyme inhibitor, resides in the ligand binding pocket with its phosphate moiety contacting the catalytically essential zinc ion through mediation of a solvent molecule. Arg47, Asp291 and Trp191 appear to be the key resi- dues for DHAP recognition in human ACMSD. Ligand binding induces a significant conformational change affecting a strictly conserved Trp–Met couple, and we propose that these residues are involved in controlling ligand admission into ACMSD. Our data may be used for the design of inhibitors with potential medical interest, and suggest a regulatory link between de novo NAD biosynthesis and glycolysis. Abbreviations ACMS, a-amino-b-carboxymuconate-e-semialdehyde; ACMSD, a-amino-b-carboxymuconate-e-semialdehyde decarboxylase; DHAP, 1,3-dihydroxyacetonephosphate; PA, picolinic acid; QA, quinolinic acid. FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6615 acid-e-semialdehyde, which possibly collapses to picoli- nic acid (PA) [2,3]. Therefore, by competing with the non-enzymatic synthesis of QA, ACMSD ultimately controls the metabolic fate of tryptophan catabolism along the kynurenine pathway, and is a medically rele- vant enzyme in light of the important roles played by QA and PA in physiological and pathological condi- tions. Indeed, QA is not only a key precursor of NAD, but also a potent neurotoxin that acts by acti- vating the N-methyl-d-aspartate subtype receptor for glutamate [4]. QA imbalance was reported to be associated with a number of neurological disorders, including a wide range of neuropsychiatric and neuro- degenerative disease states, such as epilepsy, Alzhei- mer’s and Huntington’s diseases [5]. Conversely, PA has been reported to prevent the neurotoxic effects of increased QA in the rat central nervous system, sug- gesting that a highly regulated production of these metabolites is required for normal nervous function [6]. Consistently, it has recently been shown that the enzyme is highly expressed in primary adult neurons but not in SK-N-SH neuroblastoma cells, with a per- fect correlation between the observed expression profile and the associated variation in QA and PA levels [7], and other investigations have clearly demonstrated that changes in ACMSD activity are readily reflected by serum and tissue QA levels [8]. Moreover, PA exhibits important immunomodulatory properties, being able to stimulate apoptosis [9], to efficiently interrupt the progress of human HIV-1 infection in vitro [10], and to activate macrophages in pro- inflammatory processes [11]. Most recently, abnormally high brain levels of PA have been reported in a mur- ine model of cerebral malaria, a frequently fatal com- plication of Plasmodium falciparum infection; in the same model, pharmacological reduction of PA levels was demonstrated to correlate with a better disease outcome [12,13]. ACMSD is not only present in higher eukaryotes, but also in some micro-organisms, in which the enzyme plays a key role in both the trypto- phan to QA transformation and catabolism of 2-nitro- benzoic acid [14,15]. Extensive biochemical and structural characterizations have been carried out on Pseudomonas fluorescens ACMSD (PfACMSD), lead- ing to the discovery that the enzyme is a member of the metal-dependent amidohydrolase superfamily fea- turing an (a ⁄ b) 8 TIM barrel fold [16–18]. Biochemical and structural analysis of PfACMSD led to proposal of a non-oxidative decarboxylation catalytic mecha- nism, unprecedented amongst known decarboxylases [18,19]. The gene encoding human ACMSD (hA- CMSD) was identified few years ago [20], and very recently the existence of two isoforms originating by alternative splicing was demonstrated; although comparably expressed in various organs, only the hACMSD I isoform was reported to be enzymatically active and extensively characterized [3]. hACMSD shares a high degree of sequence identity with PfACMSD (38%), with strict conservation of all resi- dues that are proposed to play a key role in catalysis and are involved in co-ordination of the catalytically essential zinc ion. As no ACMSD structure with bound substrate or inhibitor has been reported so far from any source, understanding of the ACMSD catalytic mechanism is still incomplete. In light of the reported ACMSD upregulation in the liver of streptozotocin-induced diabetic rats and the suppres- sion of such elevation following insulin injection [21,22], we decided to investigate the effect of N H CH 2 CH COOH NH 2 L-kynurenine CHO COOH COOH NH 2 Tryptophan α-amino-β-carboxymuconate-ε-semialdehyde (ACMS) α-amino-β-carboxymuconate- ε-semialdehyde decarboxylase (ACMSD) CHO COOH NH 2 α-aminomuconic acid- ε-semialdehyde (AMS) N COOH COOH Quinolinic acid non enzymatic NAD Glutaryl CoA CO2 + ATP non enzymatic N COOH Picolinic acid Fig. 1. The reaction catalyzed by hACMSD in a metabolic context. ACMS is derived from tryptophan degradation through the kynure- ine pathway, and, depending on hACMSD activity, has various metabolic destinies. Crystal structure of human ACMSD S. Garavaglia et al. 6616 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS glycolytic intermediates on the enzyme activity. Interestingly, several phosphorylated glycolytic intermediates were found to be strong inhibitors of hACMSD, of which 1,3-dihydroxyacetonephosphate (DHAP) was the most potent and was therefore selected for our structural investigation. Our results provide the first structural image of an ACMSD in a ligand-bound form, and may be used to assist the struc- ture-based rational design of enzyme inhibitors with potential medical interest. Results and Discussion Overall quality of the model The three-dimensional structure of hACMSD in com- plex with DHAP was solved by molecular replacement and refined at a resolution of 2.0 A ˚ . The monomer present in the asymmetric unit contains 332 residues out of 336, one zinc ion, one DHAP molecule, one glyc- erol molecule and a total of 216 solvent molecules. No electron density is present for the last four residues at the C-terminus. The stereochemistry of the model has been assessed using the program procheck [23]. Ninety per cent of the residues fall in the most favoured regions of the Ramachandran plot, with Asn148 and His269 falling in disallowed regions. However, for both residues, the excellent electron density map allowed us to unambiguously assign the observed conformation. Residue Pro293 was recognized as the cis conformer. Overall structure hACMSD shows a molecular architecture that closely resembles that described for PfACMSD [18], compris- ing a distorted (a ⁄ b) 8 barrel domain and a small inser- tion domain (Fig. 2). hACMSD and PfACMSD can indeed be superposed with an rmsd of 1.6 A ˚ based on 326 Ca pairs. hACMSD folds into 12 a-helices, 11 b-strands and connecting loops. Residues 14-48 form the small insertion domain that comprises a short a-helix and a three-stranded anti-parallel b-sheet; the remaining protein residues form the (a ⁄ b) 8 barrel domain and a C-terminal extension that comprises two short a-helices. Functional hACMSD was previously reported to be a monomer in solution [3]. Consistently, one molecule is present in the asymmetric unit in our crystal, although a dimer can be observed in the crys- tal lattice by applying the crystallographic two-fold axis. PfACMSD was reported to be a dimer, with subunits related by a dyad axis in the crystal, and a mixture of monomeric and dimeric forms in solution [18]. Therefore, the available structural data suggest that the minimal functional unit in the ACMSD enzyme is a monomer. and the biological significance, if any, of the loose dimer observed in the crystalline state remains to be established. The overall structural organization observed in hACMSD confirms the previ- ous assignment of the enzyme to the metal-dependent hydrolase superfamily [17,18], whose members feature by a structurally conserved TIM a ⁄ b barrel fold [24]. The significant structural conservation observed between hACMSD and PfACMSD extends to the peculiar small insertion domain, which may be consid- ered a unique trait of ACMSDs. The metal centre and the ligand binding site The hACMSD active site is located in a crevice on the protein surface at the C-terminal opening of the b-bar- rel (Fig. 2), with a Zn 2+ ion occupying the metal centre and coordinating, with a distorted trigonal bipyramidal geometry, the strictly conserved residues His6 (2.0 A ˚ ), His8 (2.1 A ˚ ), His174 (2.2 A ˚ ), Asp291 (2.2 A ˚ ) and the water molecule w1 (2.1 A ˚ ) (Fig. 3A). The DHAP binding site protrudes from the metal N-ter C-ter Fig. 2. Ribbon representation of the overall structure of hACMSD. The (a ⁄ b) 8 barrel domain is colored in light blue and the ACMSD- specific small insertion domain in blue. The DHAP molecule and the protein residues involved in metal coordination are shown as sticks; the Zn 2+ ion and the solvent molecule bridging the metal centre to the ligand are shown as magenta and cyan spheres, respectively. S. Garavaglia et al. Crystal structure of human ACMSD FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6617 centre into a small pocket delimited by residues Asp291, Trp191, Met195, Arg47 and the Phe294- Pro295-Leu296 amino acid stretch (Fig. 3A,B). The ligand binds in an extended conformation, with its phosphate moiety located in the proximity of the zinc ion and with the hydroxymethylene group pointing toward Pro295. DHAP interacts with the catalytically essential Zn 2+ through mediation of the metal-coordi- nating solvent molecule w1, which establishes two strong hydrogen bonds with the ligand O1 and O1P atoms at 2.5 and 2.8 A ˚ , respectively. Moreover, the ligand is engaged in a number of stabilizing interac- tions with the protein milieu by contacting both pro- tein residues and solvent molecules. In particular, the DHAP phosphate moiety establishes a salt bridge with Arg47 (distance of 3.0 A ˚ ), an electrostatic interaction with the Zn 2+ ion (closest distance of 4.1 A ˚ ) and an extensive network of hydrogen bonds involving a set of well-ordered solvent molecules. O1P contacts the solvent molecule w268 (2.8 A ˚ ), and O2P interacts with Trp191 (2.8 A ˚ ) and O3P with w51 (2.6 A ˚ ), which in turn contacts w119 (distance of 2.9 A ˚ ), which contrib- utes to fixing the Arg47 orientation by establishing a hydrogen bond with its NH1 atom (at 2.8 A ˚ ). The DHAP aliphatic chain is sandwiched between Trp191 and Asp291, with its carbonyl oxygen contacting, at a distance of 2.7 A ˚ , both Asp291 and the solvent mole- cule w1. Finally, the DHAP hydroxyl group is found at 3.2 A ˚ from Arg47, whose guanidinium group is held in the observed conformation by an aromatic stacking interaction with Phe297. Implications for catalysis ACMSD belongs to the metal-dependent amidohydro- lase superfamily and catalyses a non-oxidative decar- boxylation reaction through a still not completely understood mechanism. Extensive biochemical, struc- tural and spectroscopic investigations, mainly carried out on PfACMSD, led to the proposal of two possible alternative catalytic mechanisms [18], whose common feature involves formation of a tetrahedral intermedi- ate resulting from the nucleophilic attack of the metal- bound hydroxyl group onto the substrate, as observed in other members of the amidohydrolase superfamily [24,25]. However, as no structure of complexes with either the substrate, product or inhibitors had been reported, precise identification of the protein residues involved in ligand recognition and catalysis remained elusive. Although our structural data do not allow us to discriminate between the two alternative catalytic His 6 His 8 His 174 Asp 291 w 1 DHAP Arg 47 Leu 296 Trp 191 Pro 295 w 1 DHAP w 268 w 119 w 51 Met 195 Phe 294 C 1 A B Fig. 3. Close-up view of the active site in hACMSD. (A) The metal centre. The Zn 2+ ion and the coordinated solvent molecule are shown as magenta and cyan spheres, respectively. The strictly con- served protein residues forming the metal coordinative sphere and the ligand molecule are drawn as balls-and-sticks, with the DHAP phosphorous in orange. The Zn 2+ coordinative bonds are indicated by dotted lines, together with the hydrogen bonds established by DHAP with the metal-coordinating solvent molecule w1. The por- tion of the 2F o )F c electron density map covering the DHAP mole- cule is shown in blue at the 1.2 r level. (B) The DHAP binding site with crucial protein residues and solvent molecules engaged in ligand recognition and stabilization are drawn as balls-and-sticks and spheres, respectively. The major interactions established between the DHAP inhibitor and the protein milieu are indicated by dotted lines. Crystal structure of human ACMSD S. Garavaglia et al. 6618 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS mechanisms proposed for ACMSD [21], the hA- CMSD:DHAP complex represents the first structure of an ACMSD in a ligand-bound form and can be used to provide insights into catalysis. In particular, the residues involved in inhibitor binding can be suggested to be important players for recognition of the physio- logical substrate ACMS. On the basis of our structure, we propose that the strictly conserved Asp291 is a fun- damental residue for catalysis, not only because it con- tributes to Zn 2+ coordination but also because of its direct involvement in substrate binding (Fig. 3A,B). Indeed, as detailed above, Asp291 stabilizes DHAP through interaction with the inhibitor carbonyl group, and is therefore likely to demonstrate an equivalent role on ACMS by possibly recognizing its aldehyde group (Fig. 1). Our observation is in agreement with what was observed for PfACMSD, where a significant increase in the K M value was observed when the equiv- alent residue (Asp294) was mutated to alanine [16]. Another residue emerging as a key molecular determi- nant for ligand recognition in hACMSD is Arg47 (Fig. 3B). Its guanidinium group contacts both the phosphate and the hydroxyl moieties present on DHAP, and appears to be a major contributor to ACMS recognition by stabilizing interactions with the negatively charged carboxylic groups and with the aldehydic portion of the physiological substrate. A third residue of relevance for efficient ligand recogni- tion is Phe297. Indeed, in the hACMSD:DHAP struc- ture, this residue fixes the orientation of Arg47, and is ‘edge-on’ oriented with respect to the DHAP aliphatic chain with a shortest distance of 3.7 A ˚ from C1 (Fig. 3B). Such a conformation is compatible with establishment of an aromatic hydrogen bond between Phe297 and the double bonds present in ACMS. We carried out a careful comparison between the hACMSD:DHAP complex and the ligand-free form of PfACMSD after optimal structural superposition. Although no major conformational changes affecting entire domains or extended portions of the protein structure were detected, a significant change in the ori- entation of a few residues was observed (Fig. 4). Indeed, upon ligand binding, a severe conformational rearrangement takes place for the side chains of Met195 and Trp191, which move unidirectionally toward the substrate-binding pocket and become engaged with the bound ligand. Trp191 shows the most pronounced movement, which consists of a rotation around the v 2 dihedral angle of about 95°, allowing formation of a hydrogen bond with the DHAP phosphate group. This switch between two alternative conformations suggests that the Trp191–Met195 couple is the main active site gating determinant controlling ligand admission. Conclusion hACMSD appears to be an important enzyme control- ling the cellular levels of QA, PA and NAD. As a consequence, modulation of hACMSD activity could be of considerable relevance in certain therapeutic con- texts [2,26–30]. The various neurological disorders in which severe imbalance in the kynurenyne pathway is seen include cerebral malaria [31], where an elevated level of the pro-inflammatory PA has been proposed to contribute to the development of this frequently fatal clinical manifestations of the disease [12,32]. Moreover, this disease also features a significant depletion of the NAD+ level [31,33]. We propose that hACMSD inhi- bition could result in alleviation of cerebral malaria symptoms by controlling both PA and NAD levels [7,34–36]. Recently, a direct link between NAD synthesis and diabetes has been reported [37], suggesting that an increased NAD level is a desirable condition to combat the disease. Intriguingly, ACMSD was reported to be overexpressed in streptozotocin-induced diabetic rats Trp 191 w 1 DHAP Met 195 Fig. 4. Conformational changes affecting ACMSD upon ligand bind- ing. The image was obtained by optimal superposition of the hA- CMSD:DHAP and PfACMSD:ligand-free structures. Protein residues are colored in white for hACMSD and in green for PfACMSD, with the DHAP ligand in yellow; the protein portion shown by a ribbon representation refers to hACMSD, as do the amino acid numbers. The alternative conformations of the Trp–Met couple can be observed; the arrows indicate the unidirectional movement of the two protein residues upon ligand binding. The dotted line highlights the hydrogen bond established between the DHAP phosphate moiety and Trp191 in the ligand-bound form of the enzyme. S. Garavaglia et al. Crystal structure of human ACMSD FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6619 [21,22], and insulin injection was observed to suppress such elevation. Therefore, we propose that inhibition of hACMSD should be explored as a possible novel thera- peutic avenue for the treatment of diabetes. In this respect, the significant enzyme inhibition that was found to be exerted by intermediates of glycolysis (Table 1), a pathway imbalanced in diabetes, is intrigu- ing. Moreover, our structural data reveal that the enzyme active site is well suited to efficiently bind DHAP, a central glycolytic intermediate. We are there- fore tempted to speculate on a possible physiological relevance of the observed modulation of hACMSD activity by glycolytic intermediates that would imply a novel regulatory role of the enzyme in energy metabo- lism. Indeed, robust aerobic glycolysis requires signifi- cant NAD+ availability, which could be sustained by a burst of de novo dinucleotide biosynthesis through tryp- tophan degradation, an event that implies efficient AC- MSD inhibition. Therefore, hACMSD would act as a regulatory link between glycolysis and NAD synthesis. The structure of hACMSD in complex with DHAP may used for the design of potent and highly selective enzyme inhibitors that may prove to be of potential interest to reduce life-threatening complications of cerebral malaria, and as an important tool in validat- ing our proposal of hACMSD as a novel drug target for the treatment of diabetes and to investigate its proposed novel regulatory role. Experimental procedures Enzyme expression, purification and inhibition studies The expression vector pHIL-D2-ACMSDI constructed pre- viously [3] was used as a template to amplify the ACMSD gene, resulting in a C-terminal (His6) fusion protein. The amplicon was cloned into the pHIL-D2 vector and the NotI-digested construct was used to transform Pichia pas- toris GS115 cells [3]. Expression of the recombinant pro- tein was achieved as described previously [3]. Purification was performed as described previously [3] with the follow- ing modifications. The hydroxylapatite column was washed with 130 mm potassium phosphate buffer pH 7.0, 50 mm NaCl, and the recombinant protein was eluted with 300 mm potassium phosphate buffer pH 7.0, 50 mm NaCl. The active fractions were pooled and directly applied to a HisTrap HP column equilibrated in 10 mm potassium phosphate buffer pH 7.0, 100 mm NaCl. After extensive washing with the equilibration buffer, elution was per- formed with a linear gradient of imidazole from 0–0.3 m in the same buffer. The active fractions were pooled and diluted 10-fold with 50 mm potassium phosphate buffer, concentrated by ultrafiltration with a YM30 membrane (Millipore SpA, Milan, Italy) and used for the crystalliza- tion trials. Using 400 mL of yeast culture, approximately 1 mg of homogeneuos ACMSD was obtained, with a spe- cific activity (1.3 unitsÆmg )1 ) and purification index (243- fold) similar to those of the protein without the His tag, and a higher overall yield (68%) [3]. hACMSD activity was determined specrophotometrically, as described previ- ously [3]. The effect of the tested molecules on the enzyme activity was investigated in the presence of 5 lm ACMS substrate. Crystallization and structure determination Crystals of hACMSD were obtained using the vapour dif- fusion technique in hanging drop. A volume of 1 lL of res- ervoir solution containing 1 mm DHAP, 2% poly(ethylene glycol) (PEG 400), 0.1 m Na ⁄ Hepes pH 7.5, 2.0 m ammo- nium sulphate, was mixed with the same amount of a protein solution at a concentration of 12.7 mgÆmL, and equilibrated against 500 lL of the reservoir solution, at 20 °C. The crystals grew to a maximum length of 0.2 mm in approximately 1 week. The presence of DHAP was found to be essential for crystallization, and we were unable to grow crystals of hACMSD in a ligand-free form. For X-ray data collection, crystals were quickly equili- brated in a solution containing the crystallization buffer and 20% glycerol as the cryo-protectant, and flash-frozen at 100 K under a stream of liquid nitrogen. Data up to 2.0 A ˚ resolution were collected using the ID23-2 beamline of the European Synchrotron Radiation Facility (Grenoble, France). An X-ray fluorescence scan performed on the hA- CMSD crystals using the same beamline, clearly indicated the presence of a Zn metal ion bound to the enzyme. Anal- ysis of the diffraction data set allowed us to assign the crystal to the trigonal P3 2 21 or P3 1 21 space group, with cell dimensions a = b = 86.27 A ˚ and c = 92.84 A ˚ , containing one molecule per asymmetric unit with a Table 1. Effect of glycolytic intermediates on the activity of hA- CMSD. Inhibition values refer to the percentage inhibition exerted by the indicated metabolite, relative to a reaction carried out in the absence of any inhibitor (control). Experiments were performed at 37 °C in triplicate in the presence of 5 l M ACMS substrate. Metabolite Metabolite concentration (m M) Inhibition (%) Dihydroxyacetonephosphate 1 0.5 0.1 100 100 70 3-Phosphoglycerate 1 0.5 0.1 100 100 25 Glyceraldehyde-3-phosphate 1 40 Phosphoenolpyruvate 1 30 2-Phosphoglycerate 1 20 Crystal structure of human ACMSD S. Garavaglia et al. 6620 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS corresponding solvent content of 50%. Data were pro- cessed using the program mosflm [38], and the ccp 4 suite of programs [39] was used for scaling. Structure deter- mination for hACMSD was performed by means of the molecular replacement technique, using the coordi- nates of a monomer of PfACMSD as the search model (Protein Data Bank code 2HBV) [18]. The program amore [40] was used to calculate both cross-rotation and translation functions in the 10–4 A ˚ resolution range. The solution of the rotation function was used to perform the translation function calculations in both the P3 2 21 and P3 1 21 space groups. A clear solu- tion was obtained for the former only, allowing us to unambiguously assign P3 2 21 as the correct space group. The initial model was subjected to iterative cycles of crystallographic refinement using the pro- gram refmac [41], alternated with manual graphic sessions for model building using the program o [42]. Approximately 7% of the randomly chosen reflections were excluded from refinement of the structure and used for the free R-factor calculation [43]. The pro- gram arp ⁄ warp [44] was used for adding solvent mol- ecules. When the R-factor decreased to a value of 0.27 at 2.0 A ˚ resolution, inspection of the electron density map in the enzyme active site clearly revealed the pres- ence of one molecule of DHAP that was consequently manually modelled based on both the 2F o )F c and F o )F c electron density maps. The subsequent crystal- lographic refinement converged to an R-factor and a free R-factor of 0.19 and 0.24, respectively, with ideal geometry. Data collection and refinement statistics are given in Table 2. Illustrations Figures were generated by using the program pymol [45] (http://www.pymol.org). Acknowledgements The authors would like to thank Dr Franca Rossi (DiSCAFF, University of Piemonte Orientale, Novara) for critical reading of the manuscript. This work was supported by grants from the Regione Piemonte (Ricerca Scientifica Applicata 2004), Ministero dell’Is- truzione, dell’Universita ´ e della Ricerca (Bando PRIN 2007) and the Compagnia di San Paolo – IMI (Torino, Italy) in the context of the Italian Malaria Network. References 1 Moroni F (1997) Tryptophan metabolism and brain function: focus on kynurenine and other indole metabo- lites. Eur J Pharmacol 375, 87–100. 2 Mehler AH, Yano K & May EL (1964) Nicotinic acid biosynthesis: control by an enzyme that competes with a spontaneous reaction. Science 145, 817–819. 3 Pucci L, Perozzi S, Cimadamore F, Orsomando G & Raffaelli N (2007) Tissue expression and biochemical characterization of human 2-amino 3-carboxymuconate 6-semialdehyde decarboxylase, a key enzyme in tryptophan catabolism. FEBS J 274, 827–840. 4 Schwarcz R, Whetsell WO Jr & Mangano RM (1983) Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain. Science 219, 316–318. 5 Stone TW, Mackay GM, Forrest CM, Clark CJ & Darlington LG (2003) Tryptophan metabolites and brain disorders. Clin Chem Lab Med 41, 852–859. 6 Beninger RJ, Colton AM, Ingles JL, Jhamandas K & Boegman RJ (1994) Picolinic acid blocks the neurotoxic but not the neuroexcitant properties of quinolinic acid in the rat brain: evidence from turning behaviour and tyrosine hydroxylase immunohistochemistry. Neurosci- ence 61, 603–612. 7 Guillemin GJ, Cullen KM, Lim CK, Smythe GA, Garner B, Kapoor V, Takikawa O & Brew BJ (2007) Characterization of the kynurenine pathway in human neurons. J Neurosci 27, 12884–12892. 8 Shin M, Kim I, Inoue Y, Kimura S & Gonzalez FJ (2006) Regulation of mouse hepatic a-amino-b-carbo- xymuconate-e-semialdehyde decarboxylase, a key enzyme in the tryptophan–nicotinamide adenine dinu- cleotide pathway, by hepatocyte nuclear factor 4a and peroxisome proliferator-activated receptor a. Mol Pharmacol 70, 1281–1290. 9 Ogata S, Takeuchi M, Fujita H, Shibata K, Okumura K & Taguchi H (2000) Apoptosis induced by niacin-related Table 2. Data collection and refinement statistics. Parameter Value Resolution (A ˚ ) 2.0 Observations 63 794 Unique reflections 24 525 Rmerge 0.124 Multiplicity 2.6 Completeness (%) 94 Number of protein atoms 2635 Number of solvent molecules 216 Number of DHAP ⁄ Zn atoms 10 ⁄ 1 Number of glycerol atoms 6 R-factor (%) 19.3 R-free (%) 25.6 rmsd bond lengths (A ˚ ) 0.018 rmsd bond angles (°) 1.6 Mean B-factor protein (A ˚ 2 ) 15.3 Mean B-factor solvent (A ˚ 2 ) 25.2 Mean B-factor DHAP ⁄ Zn (A ˚ 2 ) 39.3 ⁄ 10.1 Mean B-factor glycerol atoms (A ˚ 2 ) 25.6 S. Garavaglia et al. Crystal structure of human ACMSD FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6621 compounds in K562 cells but not in normal human lymphocytes. Biosci Biotechnol Biochem 64, 1142–1146. 10 Fernandez-Pol JA, Klos DJ & Hamilton PD (2001) Antiviral, cytotoxic and apoptotic activities of picolinic acid on human immunodeficiency virus-1 and human herpes simplex virus-2 infected cell. Anticancer Res 21, 3773–3776. 11 Bosco MC, Rapisarda A, Reffo G, Massazza S, Pastorino S & Varesio L (2003) Macrophage activating properties of the tryptophan catabolyte picolinic acid. In The Developments in Tryptophan and Serotonin Metabolism (Allegri G, Costa CVL, Ragazzi E, Steinhart H & Varesio L eds), pp 55–65. Kluwer Academic ⁄ Plenum Publishers, New York. 12 Miu J, Ball HJ, Mellor AL & Hunt NH (2009) Effect of indoleamine dioxygenase-1 deficiency and kynurenine pathway inhibition on murine cerebral malaria. Int J Parasitol 39, 363–370. 13 Clark CJ, Mackay GM, Smythe GA, Bustamante S, Stone TW & Phillips RS (2005) Prolonged survival of a murine model of cerebral malaria by kynurenine path- way inhibition. Infect Immun 73, 5249–5251. 14 Colabroy KL & Begley TP (2005) Tryptophan cata- bolism: identification and characterization of a new degradative pathway. J Bacteriol 187, 7866–7869. 15 Muraki T, Taki M, Hasegawa Y, Iwaki H & Lau PCK (2003) Prokaryotic homologs of the eukaryotic 3-hydroxyanthranilate 3,4-dioxygenase and 2-amino- 3-carboxymuconate-6-semialdehyde decarboxylase in the 2-nitrobenzoate degradation pathway of Pseudomo- nas fluorescens strain KU-7. Appl Environ Microbiol 69, 1564–1572. 16 Li T, Walker AL, Iwaki H, Hasegawa Y & Liu A (2005) Kinetic and spectroscopic characterization of ACMSD from Pseudomonas fluorescens reveals a penta- coordinate mononuclear metallocofactor. J Am Chem Soc 127, 12282–12290. 17 Li T, Iwaki H, Fu R, Hasegawa Y, Zhang H & Liu A (2006) a-Amino-b-carboxymuconic-e-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily. Biochemistry 45, 6628–6634. 18 Martynowski D, Eyobo Y, Li T, Yang K, Liu A & Zhang H (2006) Crystal structure of a-amino-b-carbo- xymuconic-e-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylase reaction. Biochemistry 45, 10412–10421. 19 Li T, Ma JK, Holster JP, Davidson VL & Liu A (2007) Detection of transient intermediates in the metal-depen- dent nonoxidative decarboxylation catalysed by a-amino-b-carboxymuconate-e-semialdehyde decarbox- ylase. J Am Chem Soc 129, 9278–9279. 20 Fukuoka SI, Ishiguro K, Yanagihara K, Tanabe A, Egashira Y, Sanada H & Shibata K (2002) Identifica- tion and expression of a cDNA encoding human a-amino-b -carboxymuconate-e-semialdehyde decarbox- ylase (ACMSD). J Biol Chem 277, 35162–35167. 21 Tanabe A, Egashira Y, Fukuoka SI, Shibata K & Sanada H (2002) Expression of rat hepatic 2-amino- 3-carboxymuconate-6-semialdehyde decarboxylase is affected by a high protein diet and by streptozotocin- induced diabetes. J Nutr 132, 1153–1159. 22 Sasaki N, Egashira Y & Sanada H (2009) Production of l-tryptophan-derived catabolites in hepatocytes from streptozotocin-induced diabetic rats. Eur J Nutr 48, 145–153. 23 Laskowski RA, MacArthur MW, Moss DS & Thornton JM (1993) PROCHECK: a program to check the stereo- chemical quality of protein structures. J Appl Crystal- logr 26, 283–291. 24 Seibert CM & Raushel FM (2005) Structural and cata- lytic diversity within the amidohydrolase superfamily. Biochemistry 44, 6383–6391. 25 Liu A & Zhang H (2006) Transition metal-catalyzed nonoxidative decarboxylation reactions. Biochemistry 45, 10407–10411. 26 Berger F, Ramı ´ rez-Herna ´ ndez MH & Ziegler M (2004) The new life of a centenarian: signalling functions of NAD(P). Trends Biochem Sci 29, 111–118. 27 Belenky P, Bogan KL & Brenner C (2007) NAD+ metabolism in health and disease. Trends Biochem Sci 32, 12–19. 28 Magni G, Orsomando G, Raffaelli N & Ruggieri S (2008) Enzymology of mammalian NAD metabolism in health and disease. Front Biosci 13, 6135–6154. 29 Stone TW & Darlinton LG (2002) Endogenous kynure- nines as targets for drug discovery and development. Nat Rev Drug Discov 1, 609–620. 30 Schwarcz R (2004) The kynurenine pathway of trypto- phan degradation as a drug target. Curr Opin Pharma- col 4, 12–17. 31 Hunt NH, Golenser J, Chan-Ling T, Parekh S, Rae C, Potter S, Medana IM, Miu J & Ball HJ (2006) Immu- nopathogenesis of cerebral malaria. Int J Parasitol 36, 569–582. 32 Medana IM, Day NPJ, Salahifar-Sabet H, Stocker R, Smythe G, Bwanaisa L, Njobvu A, Kayira K, Turner GDH, Taylor TT et al. (2003) Metabolites of the kynurenine pathway of tryptophan metabolism in the cerebrospinal fluid of Malawian children with malaria. J Infect Dis 188, 844–849. 33 Sanni LA, Rae C, Maitland A, Stocker R & Hunt NH (2001) Is ischemia involved in the pathogenesis of murine cerebral malaria? Am J Pathol 159, 1105–1112. 34 Bender DA & Olufunwa R (1988) Utilization of tryptophan, nicotinamide and nicotinic acid as precursors for nicotinamide nucleotide synthesis in isolated rat liver cells. Br J Nutr 59, 279–287. 35 Shin M, Mori Y, Kimura A, Fujita Y, Yoshida K, Sano K & Umezawa C (1996) NAD+ biosynthesis and Crystal structure of human ACMSD S. Garavaglia et al. 6622 FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS metabolic fluxes of tryptophan in hepatocytes isolated from rats fed a clofibrate-containing diet. Biochem Pharmacol 52, 247–252. 36 Shin M, Ohnishi M, Iguchi S, Sano K & Umezawa C (1999) Peroxisome-proliferator regulates key enzymes of the tryptophan–NAD+ pathway. Toxicol Appl Pharma- col 158, 71–80. 37 Revollo JR, Ko ¨ rner A, Mills KF, Satoh A, Wang T, Garten A, Dasgupta B, Sasaki Y, Wolberger C, Town- send RR et al. (2007) Nampt ⁄ PBEF ⁄ Visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme. Cell Metab 6, 363–367. 38 Leslie AG (1999) Integration of macromolecular diffrac- tion data. Acta Crystallogr D 55, 1696–1702. 39 Collaborative Computational Project Number 4 (1994) The CCP4 suite: programs for protein crystallography. Acta Crystallogr D 50, 760–763. 40 Navaza J (2001) Implementation of molecular replace- ment in AMoRe. Acta Crystallogr D 57, 1367–1372. 41 Murshudov GN, Vagin AA & Dodson EJ (1997) Refine- ment of macromolecular structures by the maximum- likelihood method. Acta Crystallogr D 53, 240–255. 42 Jones TA, Zou JY, Cowan SW & Kjeldgaard M (1991) Improved methods for building protein models in elec- tron density maps and the location of errors in these models. Acta Crystallogr A 47, 110–119. 43 Bru ¨ nger AT (1993) Assessment of phase accuracy by cross validation: the free R value. Methods and applica- tions. Acta Crystallogr D 49, 24–36. 44 Perrakis A, Morris R & Lamzin VS (1999) Automated protein model building combined with iterative struc- ture refinement. Nat Struct Biol 6, 458–463. 45 DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, Palo Alto, CA. S. Garavaglia et al. Crystal structure of human ACMSD FEBS Journal 276 (2009) 6615–6623 ª 2009 The Authors Journal compilation ª 2009 FEBS 6623 . (QA) and NAD homeostasis. Indeed, the enzyme stands at a branch point of the tryptophan to NAD pathway, and deter- mines the final fate of the amino acid,. Fukuoka SI, Ishiguro K, Yanagihara K, Tanabe A, Egashira Y, Sanada H & Shibata K (2002) Identifica- tion and expression of a cDNA encoding human a- amino-b -carboxymuconate-e-semialdehyde