Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis Ilaria Marinoni 1 , Simona Nonnis 2 , Carmine Monteferrante 1 , Peter Heathcote 3 , Elisabeth Ha ¨ rtig 4 , Lars H. Bo ¨ ttger 5 , Alfred X. Trautwein 5 , Armando Negri 2 , Alessandra M. Albertini 1 and Gabriella Tedeschi 2 1 Department of Genetics and Microbiology, University of Pavia, Italy 2 D.I.P.A.V., Section of Biochemistry, University of Milano, Italy 3 School of Biological and Chemical Sciences, Queen Mary College, University of London, UK 4 Institute of Microbiology, Technical University of Braunschweig, Germany 5 Institute of Physics, University of Lu ¨ beck, Germany NAD is a ubiquitous and essential molecule in all living organisms. In addition to its well-established role in redox biochemistry and energetic metabolism, NAD can function as a signaling molecule in a variety of cellular processes [1]. In eubacteria, NAD is pro- duced by a de novo pathway or starting from pre- formed nicotinic acid. Quinolinic acid is the precursor for the de novo pathway; in most eukaryotes, it is pro- duced via degradation of tryptophan, whereas in many eubacteria, including several pathogens, it is synthe- sized from l-aspartate and dihydroxyacetone phos- phate (DHAP). This reaction involves the so-called quinolinate synthase complex: the first enzyme, l-aspartate oxidase (NadB, EC 1.4.3.16), encoded by the gene nadB, catalyzes the oxidation of l-aspartate to iminoaspartate; the second enzyme, quinolinate syn- thase (NadA), is encoded by the gene nadA and cata- lyzes the condensation between iminoaspartate and DHAP, resulting in quinolinic acid production (Scheme 1) [2]. Quinolinic acid is then converted to Keywords L-aspartate oxidase; NAD biosynthesis; NadA; NadB; quinolinate synthase Correspondence G. Tedeschi, D.I.P.A.V., Section of Biochemistry, University of Milano, Via Celoria 10, 20133 Milano, Italy. Fax: +39 02 50318123 Tel: +39 02 50318127 E-mail: gabriella.tedeschi@unimi.it (Received 4 July 2008, revised 1 August 2008, accepted 12 August 2008) doi:10.1111/j.1742-4658.2008.06641.x NAD is an important cofactor and essential molecule in all living organ- isms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacil- lus subtilis, one of the best studied bacteria and a model organism for low- GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe–4S] 2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe–4S] 2+ cluster is coordi- nated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new non- canonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli. Abbreviations DHAP, dihydroxyacetone phosphate; GST, glutathione S-transferase; GST–NadA, quinolinate synthase fused to glutathione S-transferase (GST) at its N-terminus; IPTG, isopropyl thio-b- D-galactoside; NadA, quinolinate synthase; NadA–His, quinolinate synthase with a His 6 -tag at the N-terminus; NadB, L-aspartate oxidase. 5090 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS nicotinic acid and, finally, to NAD by a biosynthetic pathway common to all organisms. As NadA and NadB are absent in mammals, they are considered to be ideal targets for the development of novel prophy- lactic and therapeutic agents [3]. Moreover, very recently, Pruinier et al. [4] reported that the pathogenic bacterium Shigella is a nicotinic acid auxotroph, unable to synthesize NAD via the de novo pathway, due to nadA and nadB gene mutations. When the func- tionality of nadA ⁄ B in Shigella was restored, a consis- tent loss of virulence and inability to invade host cells were observed. On the basis of this result, they defined NadA and NadB as antivirulence loci. Besides being important in bacteria, NadA and NadB analogs seem to be involved in NAD biosynthe- sis also in plants. Many experimental findings, together with the apparent absence of genes encoding enzymes involved in other possible routes to quinolinate, sug- gest that several plants may obtain this key precursor via the aspartate pathway, like many bacteria [5–7]. Therefore, because of the growing amount of evi- dence indicating the importance in several organisms of de novo NAD biosynthesis through the reaction catalyzed by NadA and NadB, it is of the utmost importance to gain a thorough knowledge of the bio- chemical and structural properties of these two enzymes. The gene nadB is present in several microorganisms and in plants, but the protein has been purified only from Escherichia coli, Pyrococcus horikoshii and Sulfol- obus tokadaii, and characterized from a biochemical and structural point of view only from E. coli and S. tokadaii [8–17]. It is a flavoprotein containing 1 mol of noncovalently bound FAD ⁄ mol of protein. This enzyme presents several peculiarities that distinguish it from all other flavo-oxidases: (a) in vitro, it is able to use different electron acceptors such as oxygen, fuma- rate, cytochrome c and quinones [9], suggesting that it is involved in NAD biosynthesis in anaerobic as well as aerobic conditions; and (b) the primary and tertiary structures are not similar to those of other flavo-oxid- ases, but to those of the flavoprotein subunit of the succinate dehydrogenase ⁄ fumarate reductase class of enzymes. As a consequence, NadB shares with these proteins most of the active site features, including the presence of an arginine playing an acid–base role in catalysis [11–15]. Accordingly, NadB can reduce fuma- rate, but it is unique in that it is able to stereospecifi- cally oxidize l-aspartate and is unable to oxidize succinate. Interestingly, in 2003, Yang et al. [18] described another enzyme, from Thermotoga maritima, that is involved in the de novo biosynthesis of NAD and that plays the same role as NadB, although it does not share any recognizable sequence similarity to NadB. It is described as NADP-dependent l-aspartate dehydro- genase, and is strictly specific for l-aspartate. This enzyme produces iminoaspartate, which is then con- verted to quinolinate through the condensation with DHAP catalyzed by NadA. The second enzyme involved in the de novo biosyn- thesis of NAD, NadA, is extremely sensitive to oxygen; therefore, it has been poorly characterized so far, and very little is known regarding its biochemical and structural properties. The enzyme has only been puri- fied from E. coli [19–21] and P. horikoshii [22]. Recent studies on the enzyme from E. coli have demonstrated that the protein harbors a [4Fe–4S] 2+ cluster [20,21] that, as it is very sensitive to oxygen, probably explains why NadA is identified as the site of oxygen poisoning of NAD synthesis in anaerobic bacteria [23]. The 3D structure has been obtained for the enzyme from P. horikoshii [22]. The protein shows a triangular architecture in which conserved amino acids determine three structurally homologous domains. Unfortunately, the structure lacks any data on the [Fe–S] center, and the three surface loops that contain two highly conserved cysteine residues are disordered. Moreover, Scheme 1. Reaction catalyzed by the ‘quinolinate synthase complex’. The first enzyme, NadB, catalyzes the oxidation of L-aspartate to iminoaspartate using either oxygen or fumarate as electron acceptor for FAD reoxidation; the second enzyme, NadA, catalyzes the conden- sation between iminoaspartate and DHAP, resulting in quinolinic acid production. I. Marinoni et al. NadA and NadB from B. subtilis FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5091 the canonical binding motif for [4Fe–4S] 2+ clusters (CXXCXXC) that is found in the C-terminal regions of most quinolinate synthases from bacteria, including E. coli [19–21], is absent in NadA from P. horikoshii, and in this case the cofactors remain to be identified [22]. The absence of the consensus sequence for the binding of a [4Fe–4S] 2+ cluster is observed also in NadA from several plants. On the other hand, very recently Murthy et al. [7] described a new SufE-like protein from Arabidopsis thaliana chloroplasts that contains two domains, one SufE-like domain and one with similarity to the bacterial NadA carrying a highly oxygen-sensitive [4Fe–4S] 2+ cluster. Therefore, two important areas have to be clarified: (a) the nature of the cofactor for quinolinate synthase, in particular for NadA proteins that do not contain a canonical binding motif for a [4Fe–4S] 2+ cluster; and (b) the identifica- tion of the residues involved in the binding of the [4Fe–4S] 2+ cluster, if present. In an attempt to resolve some of these issues, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model for low-GC Gram-positive bacte- ria, including pathogens. Our data add new informa- tion regarding the NadA cofactor and the interaction between NadA and NadB. In particular, it is demon- strated that the cofactor for NadA from B. subtilis is a [4Fe–4S] 2+ cluster, even though the sequence does not show a canonical binding motif. Moreover, for the first time, the cysteines involved in the cluster binding are identified. Taken together, our data suggest that in NadA from B. subtilis, the [4Fe–4S] 2+ cluster is coor- dinated by three strictly conserved cysteine residues (Cys110, Cys230, and Cys320). Thus, NadA presents a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different sources. More- over, the results show for the first time that the inter- action between NadA and NadB is not species-specific between the proteins from B. subtilis and E. coli. Results and Discussion NadA cloning and protein purification and characterization In order to optimize the heterologous production and purification of B. subtilis NadA, several expression vectors with different tags were utilized: NadA with a His 6 -tag at the N-terminus, NadA with a His 6 -tag at the C-terminus (NadA–His), and NadA fused to gluta- thione S-transferase (GST) at its N-terminus (GST– NadA). The best results in terms of soluble protein yield were obtained by cloning the nadA gene in pET28-a with the His-tag at the C-terminal region. Upon purification in a glove box under anaerobic con- ditions, a soluble pure protein, brown in color, was obtained with a yield of 10 mg of pure protein from 1L of E. coli culture expressing B. subtilis NadA (Fig. 1A). Therefore, this protein was utilized for further studies. As determined by gel filtration, it is a trimer of 124 kDa (expected molecular mass for the monomer 41 kDa) under both aerobic and anaerobic conditions (data not shown). To evaluate its enzymatic activity, quinolinate for- mation was measured by a discontinuous enzymatic assay that couples the production of iminoaspartate by NadB with the condensation between DHAP and iminoaspartate to form quinolinic acid catalyzed by NadA [19] (Scheme 1). As described below, NadB is able to use both molecular oxygen and fumarate as electron acceptors for FAD reoxidation. Therefore, to better evaluate NadA activity, the assays were per- formed under aerobic and anaerobic conditions (in the presence of fumarate), using recombinant B. subtilis NadA plus B. subtilis NadB, overexpressed and puri- fied as detailed below. Different concentrations of NadA, NadB and fumarate (under anaerobic condi- tions) were utilized in order to set up a suitable assay to be used to check NadA activity. The data showed that: (a) the assay is linear up to 0.25 mg of NadA; (b) 10 lg of NadB is the lowest amount suitable to measure NadA activity; and (c) under anaerobic condi- tions, NadA activity becomes independent of fumarate concentration, starting from 1 mm fumarate, but decreases at concentrations higher than 2 mm fuma- rate, due to inhibition of NadB by fumarate [9]. There- fore, to evaluate quinolinate formation, the assay routinely used contained 70 lg of NadA, 30 lgof NadB and 1 mm fumarate under anaerobic conditions. AB Fig. 1. Production and purification of NadA from Bacillus subtilis. (A) 11% SDS ⁄ PAGE of NadA–His before and after purification in a glove box. Std, molecular markers; P, pellet; S, soluble fraction; NadA–His, purified protein. (B) Visible absorption spectrum of NadA–His purified under anaerobic conditions ( _______ ) and after 2 h of exposure to air (- - -). NadA and NadB from B. subtilis I. Marinoni et al. 5092 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS An apparent K m of 0.36 ± 0.05 mm was calculated for DHAP at 25 °C, using oxygen as electron acceptor. The specific activity of NadA from B. subtilis was 0.05 ± 0.01 lmolÆmin )1 Æmg )1 in the presence of fuma- rate as electron acceptor for NadB, and 0.027 ± 0.01 lmolÆmin )1 Æmg )1 using oxygen to reoxidize NadB. These values were more than two times higher than that reported for NadA from E. coli (0.015 lmolÆmin )1 Æmg )1 using fumarate) [20,21], and comparable to the results described for SufE3 purified from A. thaliana, which catalyzes the for- mation of quinolinate with a specific activity of 0.05 lmolÆmin )1 Æmg )1 using fumarate as electron acceptor for NadB [7]. Similar data were obtained if NadA without tags or GST–NadA was used in the assay mixture instead of NadA–His, ruling out the possibility that the presence of a tag at either the N-terminus or C-terminus had any effect on the enzymatic activity. NadA from B. subtilis contains an oxygen-labile [4Fe–4S] 2+ cluster as a cofactor Figure 1B shows the absorbance spectrum of NadA from B. subtilis purified under anaerobic conditions. The shoulder at 420 nm in the spectrum suggests the presence of an [Fe–S] cluster in the protein. This clus- ter appears to be oxygen-sensitive, because absorption in the visible region was altered after exposure to air, with a progressive decrease of the absorption in the 420 nm region (Fig. 1B). Using the protein purified in the glove box, it was possible to determine that the protein contained 3.8 ± 0.2 mol iron ⁄ mol NadA and 3.3 ± 0.01 mol inorganic sulfide ⁄ mol protein, suggest- ing the presence of one [4Fe–4S] 2+ cluster per mono- mer of NadA. In accordance with this finding, complete loss of activity was detected for NadA from B. subtilis purified under anaerobic conditions and exposed to oxygen overnight, as the cluster integrity is compromised in such conditions. These results are in agreement with the data reported for NadA from E. coli and A. thaliana, which contain one highly oxy- gen-sensitive [4Fe–4S] 2+ cluster per monomer of NadA [7,20,21]. The data are in keeping with the hypothesis proposed by Sun & Setlow [24], who suggested that NadA from B. subtilis may contain an [Fe–S] cluster, on the basis of the observation that, like E. coli, B. subtilis iscS ) strains are auxotrophic for nicotinic acid and are unable to synthesize NAD de novo. Further characterization of the [Fe–S] cofactor was performed by Mo ¨ ssbauer and EPR spectroscopy (Figs 2 and 3, respectively). The Mo ¨ ssbauer spectrum of an NadA sample, recorded at 77 K, is shown in Fig. 2A. At first glance, it is appropriate to fit this spectrum with one quadrupole doublet, representing 100% of the iron in the NadA sample. The resulting fit parameters (isomer shift d = 0.44 mmÆs )1 , quadru- pole splitting DE Q = 1.05 mmÆs )1 , and line width G = 0.48 mmÆs )1 ) are characteristic for [4Fe–4S] 2+ clusters. The [4Fe–4S] 2+ clusters in other biological systems exhibit similar Mo ¨ ssbauer parameters [21,25,26]. The Mo ¨ ssbauer spectrum of NadA that was exposed to air at room temperature (for 30 min), mea- sured at 77 K, reveals that the [4Fe–4S] 2+ clusters are oxygen-sensitive and are decomposed (Fig. 2B). About 55% of the iron in that spectrum still repre- sents [4Fe–4S] 2+ clusters; the remaining 45% of the absorption pattern appears as a quadrupole doublet with Mo ¨ ssbauer parameters (d = 0.27 mmÆs )1 , DE Q = 0.53 mmÆs )1 and G = 0.35 mm Æs )1 ) that are A B C Fig. 2. NadA contains a [4Fe–4S] cluster: Mo ¨ ssbauer spectra mea- sured at 77 K. (A) The quadrupole doublet represents [4Fe–4S] 2+ clusters. (B) NadA exposed to air for 30 min. The two quadrupole doublets represent [4Fe–4S] 2+ clusters (dashed line) and, in addi- tion, high-spin (S =5⁄ 2) tetrahedral-sulfur-coordinated iron sites (dotted line) (see text). (C) Reanalysis of the measured spectrum from (A) with two quadrupole doublets representing the 3 : 1 bind- ing motif of the [4Fe–4S] 2+ clusters (see text). The solid line is the envelope of the dashed and dotted lines in (B) and (C). I. Marinoni et al. NadA and NadB from B. subtilis FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5093 characteristic for high-spin (S =5⁄ 2) tetrahedral-sul- fur-coordinated iron sites as observed in [2Fe–2S] 2+ clusters [21,27]. Oxygen sensitivity has been observed for [4Fe–4S] 2+ clusters in other proteins, where this sensitivity leads to partial or total degradation of these clusters. Such proteins have in common that their [4Fe–4S] 2+ clusters are ligated by only three cysteines, whereas the fourth iron is coordinated by a nonprotein ligand [28,29]. It was thus tempting to reanalyze the Mo ¨ ssbauer spectrum of NadA (which was not exposed to air), but now assuming two different kinds of iron sites in the [4Fe–4S] 2+ cluster, which is coordinated with a 3 : 1 ratio of cysteine to noncysteine. This situa- tion requires two quadrupole doublets with an area ratio of 3 : 1, instead of one doublet only. A tetrahe- dral-coordinated Fe 2.5+ site, with the cysteine ligand replaced by a nonsulfur ligand, i.e. nitrogen or oxygen, is expected to exhibit an increase of isomer shift by around 0.05–0.1 mmÆs )1 in comparison with the three tetrahedral-sulfur-coordinated Fe 2.5+ sites. Visualiza- tion of this specific 3 : 1 binding motif in a Mo ¨ ssbauer spectrum was provided before for the [4Fe–4S] 2+ clus- ters in the ferredoxin of the anaerobic ribonucleotide reductase from E. coli [30], in the ferredoxin from the hyperthermophilic archeon Pyrococcus furiosus [31], in the (E)-4-hydroxy-3-methylbut-2-enyl diphosphate syn- thase from A. thaliana [25], and in the radical S-adeno- sylmethionine enzyme coproporphyrinogen III oxidase HemN [26]. A corresponding fit of the Mo ¨ ssbauer spectrum of NadA using two quadrupole doublets (Fig. 2C) yields the following: doublet I (dashed line; the relative absorption area 75% was fixed in the fit; d I = 0.42 mmÆs )1 , DE QI = 1.05 mmÆs )1 , G I = 0.43 mmÆs )1 ) represents tetrahedral-sulfur-coordi- nated Fe 2.5+ sites, and doublet II (dotted line; the rela- tive area 25% was fixed in the fit; d II = 0.52 mmÆs )1 , DE QII = 1.09 mmÆs )1 , G II = 0.42 mmÆs )1 ) represents the tetrahedral-coordinated Fe 2.5+ site with the cyste- ine ligand replaced by a noncysteine ligand. The Mo ¨ ssbauer parameters of the two doublets are in rea- sonable agreement with those reported for [4Fe–4S] 2+ clusters in other proteins with this specific 3 : 1 bind- ing motif [25,26,30,31]. The EPR spectrum of the ‘as isolated’ protein (not presented) showed a trace contribution from a [3Fe–4S] center at g = 2.031 and a relatively minor amount of free iron at g = 4.2. However, the spec- trum did contain a relatively significant contribution from Cu 2+ , so the ‘as isolated’ spectrum was sub- tracted from the spectra of the reduced samples to pre- vent this baseline signal distorting the EPR spectra of the [Fe–S] center at low fields. The difference (reduced ) oxidized) EPR spectra of the NadA protein reduced at pH 8 and pH 10 are presented in Fig. 3. The sharp derivative signal around g = 2.00 arises from the radical of methyl viologen, which was added to the samples as a redox mediator. The EPR spec- trum of the reduced NadA protein produces an EPR spectrum in the pH 10 sample at 15 K, which is typical of a [4Fe–4S] 1+ center with g 1 = 2.054 and g 2,3 = 1.932. Interestingly, the sample at pH 8 indi- cates that the [Fe–S] center exists in two slightly differ- ent forms, with this difference being indicated by a split in the high field feature with features at g = 1.94 and g = 1.89. This could be caused by slight differ- ences in folding of the protein, or a charged residue close to the [Fe–S] center that has a pK a close to that of the sample at pH 8 so that it is only charged in a fraction of the samples (about 50%). The shift to pH 10 clearly favors the g = 1.93 ⁄ g = 1.94 fea- ture ⁄ conformation ⁄ state. Given that it is thought from studies reported in this article that the fourth ligand to this [4Fe–4S] 2+ center is not a cysteine, it is tempting to speculate that the two different forms of the [Fe–S] cluster detected at pH 8 may reflect differences in the fourth noncysteine ligand. We estimate that 70–80% of the maximal [Fe–S] content of these samples is contributing to the EPR spectra recorded. Identification of [Fe–S] cluster-binding residues of NadA Recent studies on NadA from E. coli and on SufE3 from A. thaliana demonstrated that these enzymes harbor a [4Fe–4S] 2+ cluster that is essential for the A B Fig. 3. NadA contains a [4Fe–4S] cluster: EPR spectra of NadA reduced at pH 8 and pH 10. NadA was reduced with sodium dithio- nite and methyl viologen, as described in Experimental procedures. The two spectra presented represent the difference between the reduced sample and the unreduced control. The experimental con- ditions for acquisition of the spectrum were: microwave power, 2 mW; modulation amplitude, 0.1 mT; temperature, 15 K. NadA and NadB from B. subtilis I. Marinoni et al. 5094 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS activity [20,21], but no information is available regard- ing the residues involved in binding of the [Fe–S] clus- ter. The canonical binding motif for the [4Fe–4S] 2+ cluster CXXCXXCX was found in many quinolinate synthases from bacteria (including E. coli and Synecho- cystis), and it was proposed that the cysteines of this pattern are involved in the binding of the cluster. However, as shown in Fig. 4, NadA from B. subtilis lacks this typical cysteine motif and shows a different arrangement of conserved cysteines from that in E. coli NadA. The same observation was reported for the homologous sequence of the bacterial quinolinate synthase found in chloroplasts of A. thaliana, Oryza sativa, poplar, medicago and other plant species, and for the enzyme from P. horikoshii, an anaerobic hyperthermophilic archaeon whose crystal structure was solved in 2005 [22]. On the other hand, all the res- idues involved in the binding of malate in the crystal structure of NadA from P. horikoshii [22] are strictly conserved in all the NadA sequences reported so far in the data banks. Figure 4 reports only few of them, for reasons of clarity. The sequence alignment analyses suggest that all quinolinate synthases may share the unique triangular architecture described for the protein from P. horikoshii. Unfortunately, this partial structure lacks the [Fe–S] cluster, and the three surface loops that contain two highly conserved cysteine residues are disordered. Therefore, the question of which residues are important for the binding of the [Fe–S] cluster is still unanswered. The multiple alignment shows that three cysteines are strictly conserved in all the plant and bacterial sequences reported so far, and thus are very good candidates as iron ligands (Fig. 4). Muta- genesis studies on NadA from B. subtilis allowed us to substantiate this hypothesis. B. subtilis nadA encodes six cysteine residues. Three of them are not shared with all the proteins represented in Fig. 4 but are well conserved in the MF_00569 family, one of the three NadA families of the HAMAP database [32], which comprises mainly proteins from Gram-positive bacteria and some archeans of the genus Halobacterium. This family is very distinct from the other two: the MF_0567 family (including E. coli NadA), comprising proteins mainly from Gram-negative bacteria, and the MF_00568 family, which contains NadA proteins from bacteria and archeans (e.g. from Mycobacterium tuber- culosis and P. horikoshii) and plastids. In contrast, Cys110, Cys230 and Cys320 in NadA from B. subtilis are strictly conserved in all the NadA sequences reported so far. Single point mutations to serine were carried out for all the six residues (Cys82, Cys110, Cys230, Cys259, Cys318 and Cys320), and the mutant enzymes purified were tested for enzymatic activity and iron content (Table 1). In total, six single NadA mutants and a double mutant carrying the C318S ⁄ C320S substitution were generated. The yield and stability of all mutated proteins were comparable to the those obtained for the wild-type NadA. The enzymes with mutations of nonconserved residues, C82S and C259S, showed the same activity, the same iron content and the same spectral properties as the wild-type. In contrast, the enzymes with mutations at conserved residues, C110S and C230S, were almost colorless and inactive, indicating that these residues are absolutely vital for [Fe–S] cluster formation (Table 1). The third residue conserved in all the NadA sequences is Cys320. The C320S mutant was inactive but was still able to bind 1.5 iron atoms ⁄ mol protein, probably because, in the absence of Cys320, Cys318 may play an ancillary role in iron binding. In contrast, the C318S mutant was fully active and was able to bind 3.1 iron atoms ⁄ mol protein, unlike the double mutant C318S ⁄ C320S, which was colorless and inac- tive. Taken together, the data suggest that in NadA from B. subtilis the [4Fe–4S] 2+ cluster is coordinated by three highly conserved cysteine residues (Cys110, Cys230, and Cys320). The results of site-directed muta- genesis are in agreement with the fit of the Mo ¨ ssbauer and EPR spectra reported above, suggesting that NadA presents a new noncanonical binding motif that, we propose, may be common to other quinolinate synthases from different sources. In vivo Nic phenotype verification of NadA mutants As previously reported [24], mutations in the nadBCA or in the divergent iscS ⁄ nifS operons confer on B. sub- tilis a Nic ) phenotype (nicotinic acid requirement in minimal medium, due to impairment of the de novo pathway). To verify the phenotype conferred in vivo by the cysteine to serine substitutions described in the pre- vious section, we tested the ability to grow in minimal medium with and without nicotinic acid of the six B. subtilis NadA single mutants C82S, C110S, C230S, C259S, C318S, and C320S, and, as a negative control, of the DnadA mutant obtained with the allelic switch protocol described in Experimental procedures [33]. The phenotype of isolated clones that each bear a single cysteine to serine mutation is shown in Fig. 5. After 24 h of growth in aerobic conditions on minimal medium with glucose (0.5%) and tryptophan (50 lgÆmL )1 ), the C110S, C230S, C320S and C318S mutants showed, in the absence of nicotinic acid, the same growth impairment as a DnadA strain. The addi- tion of 0.5–50 lgÆmL )1 nicotinic acid was sufficient to I. Marinoni et al. NadA and NadB from B. subtilis FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5095 promote normal growth. The C110S, C230S and C320S mutations thus confer on B. subtilis a Nic ) phe- notype, confirming the observations made in vitro with the purified mutant enzymes. In contrast, the absence of involvement of Cys318 in NadA activity was not confirmed by the in vivo study: this cysteine must play NADA_ECOLI MSVMFDPDTAIYPFPPKPTPLSIDEKAYYREKIKRLLKERNAVMVAHYYTDPEIQQLAEETGGCI SDSLEMARFGAKHP ASTLLVAGVRFMGETAKILSPEK 102 NADA_SALTY MSVMFDPQAAIYPFPPKPTPLNDDEKQFYREKIKRLLKERNAVMVAHYYTDPEIQQLAEETGGCI SDSLEMARFGTKHA ASTLLVAGVRFMGETAKILSPEK 102 NADA_BACSU MSILDVIKQSNDMMPESYKELSRKDMETRVAAIKKKFGSRLFIPGHHYQKDEVIQFADQTG DSLQLAQVAEKNKE ADYIVFCGVHFMAETADMLTSEQ 98 NADA_HELPY MPTDNDLKAAILELLRDLDVLLVAHFYQKDEIVELAHYTG DSLELAKIASQS-D KNLIVFCGVHFMGESVKALAFDK 76 NADA_METTH MLNQLQRDILRLKKEKNAIILAHNYQSREIQEIADFKG DSLELCIEASRIEG KDIVVFCGVDFMAETAYILNPDK 75 NADA_METJA MSMDIVERINKLKEEKNAVILAHNYQPKEIQKIADFLG DSLELCIKAKETD ADIIVFCGVDFMGESAKILNPEK 74 NADA_PYRHO MDLVEEILRLKEERNAIILAHNYQLPEVQDIADFIG DSLELARRATRVD ADVIVFAGVDFMAETAKILNPDK 72 NADA_AQUAE MVQLALKEEKELTKEEIKELQKEVRRLAKEKNAVILAHYYQRPEVQDIADFVG DSLELARKASQTD ADIIVFCGVRFMCETAKIVNPEK 89 NADA_SYNY3 MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG DSLGLSQQAASTD ADVIVFAGVHFMAETAKILNPHK 85 NADA_SYNEC MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG DSLGLSQQAASTD ADVIVFAGVHFMAETAKILNPHK 85 NADA_CYAPA MSIFLKNKQFENITSQEQTKNNYKQLINDIQTLKKDLNAIILAHYYQEPDIQDVADFLG DSLGLAREAAKTN ADIIVFAGVHFMAETAKILNPEK 95 NADA_EHRCR MKELDVIT LLQEIRHLAQESNAVILAHYYQDSEIQDIADFIG DSLELSRKAATTT ADVIVFCGVYFMAEVAKIINPAK 78 NADA_MYCLE MTVLNGMEPLAGDMTVVIAGITDSPVGYAGVDGDEQWATEIRRLTRLRGATVLAHNYQLPAIQDIADYVG DSLALSRIAAEVP EETIVFCGVHFMAETAKILSPNK 106 NADA_MYCTU MTVLNRTDTLVDELT ADITNTPLGYGGVDGDERWAAEIRRLAHLRGATVLAHNYQLPAIQDVADHVG DSLALSRVAAEAP EDTIVFCGVHFMAETAKILSPHK 103 NADA_ATHAL VPSFEPFPSLVLTAHGIEAKGSFAQAQAKYLFPEESRVEELVNVLKEKKIGVVAHFYMDPEVQGVLTAAQKHWPHISISDSLVMADSAVTMAKAGCQFITVLGVDFMSENVRAILDQAGF 120 NADA_OSATI MFLSPNESKTSELVKSLREKKIGIVAHFYMDPEVQGILTASKKHWPHIHISDSLVMADSAVKMAEAGCEYITVLGVDFMSENVRAILDQAGY 92 : .* * : . *** :. . . : . ** ** * . : NADA_ECOLI TILMPT-LQAEC SLDLGCPVEEFNAFCDAHPDRT VVVYANTSAAVKARAD WVVTSSIAVELIDHL DSLGEKIIWAPDKHLGRYVQKQTGG 191 NADA_SALTY TILMPT-LAAECSLDLGCPIDEFSAFCDAHPDRT VVVYANTSAAVKARAD WVVTSSIAVELIEHL DSLGEKIIWAPDRHLGNYVQKQTGA 191 NADA_BACSU QTVVLPDMRAGCSMADMADMQQTNRAWKKLQHIFGDTIIPLTYVNSTAEIKAFVGKHG-GATVTSSNAKKVLEWA FTQKKRILFLPDQHLGRNTAYDLGIALEDMAVWDPMKDEL 212 NADA_HELPY QVIMP KLSCCSMARMIDSHYYDRSVHLLKECGVKEFYPITYINSNAEVKAKVAKDD-GVVCTSRNASKIFNHA LKQNKKIFFLPDKCLGENLALENGLKSAILGANS 182 NADA_METTH KILIPD-RGAECPMAHMLSAEDVRMARKRYPDAA VVLYVNTLAEAKAEAD ILCTSANAVRVVES LDEDLVLFGPDRNLAWYVQEHT 160 NADA_METJA KVLMPEIEGTQCPMAHQLPPEIIKKYRELYPEAP LVVYVNTTAETKALAD ITCTSANADRVVNS LDADTVLFGPDNNLAYYVQKRT 160 NADA_PYRHO VVLIPS-REATCAMANMLKVEHILEAKRKYPNAP VVLYVNSTAEAKAYAD VTVTSANAVEVVKK LDSDVVIFGPDKNLAHYVAKMT 157 NADA_AQUAE KVLHPN-PESGCPMADMITAKQVRELREKHPDAE FVAYINTTADVKAEVD ICVTSANAPKIIKK LEAKKIVFLPDQALGNWVAKQV 174 NADA_SYNY3 LVLLPD-LEAGCSLADSCPPREFAEFKQRHPDHL VISYINCTAEIKALSD IICTSSNAVKIVQQ LPPDQKIIFAPDRNLGRYVMEQTGR 173 NADA_SYNEC LVLLPD-LEAGCSLADSCPPREFAEFKQRHPDHL VISYINCTAEIKALSD IICTSSNAVKIVQQ LPPDQKIIFAPDRNLGRYVMEQTGR 173 NADA_CYAPA MVLLPD-LNAGCSLADSCPPEIFSEFKKAHSDHL VISYINCSASIKAMSD IICTSANAVDIVNK IPLTQPILFAPDQNLGRYVISKTGR 183 NADA_EHRCR KVLLPD-LNAGCSLADSCDAESFKKFRELHKDCV SITYINSLAEVKAYSD IICTSSSAEKIIRQ IPEEKQILFAPDKFLGAFLEKKTNR 166 NADA_MYCLE TVLIPD-QRAGCSLADSITPDELCAWKDEHPGAA VVSYVNTTAEVKALTD ICCTSSNAVDVVES IDPSREVLFCPDQFLGAHVRRVTGRK 195 NADA_MYCTU TVLIPD-QRAGC SLADSITPDELRAWKDEHPGAV VVSYVNTTAAVKALTD ICCTSSNAVDVVAS IDPDREVLFCPDQFLGAHVRRVTGRK 192 NADA_ATHAL EKVGVYRMSDETIGCSLADAASAPAYLNYLEAASRSPPS LHVVYINTSLETKAFAHELVPTITCTSSNVVQTILQAFAQMPELTVWYGPDSYMGANIVKLFQQMTLMTNEEIANIHPK 238 NADA_OSATI SKVGVYRMSSDQIGCSLADAASSSAYTHFLKEASRSPPS LHVIYINTSLETKAHAHELVPTITCTSSNVVATILQAFAQIPGLNVWYGPDSYMGANIADLFQRMAVMSDEEIAEVHPS 210 *.: : * * ** ** . . : : ** :. NADA_ECOLI DILCWQGACIVHDEFKTQALTRLQEEYPDAAILVHPES PQAIVDMADAVGSTSQLIAAAK TLPH-QRLIVATDRGIFYKMQQAVPDKE 278 NADA_SALTY DVLCWQGACIVHDEFKTQALTRLKKIYPDAALLVHPES PQSIVEMADAVGSTSQLIKAAK TLPH-RQLIVATDRGIFYKMQQAVPEKE 278 NADA_BACSU V AESGHTNVKVILWKGHCSVHEKFTTKNIHDMRERDPDIQIIVHPEC SHEVVTLSDDNGSTKYIIDTIN QAPAGSKWAIGTEMNLVQRIIHEHPDK- 308 NADA_HELPY QEEIKNADVVCYNGFCSVHQLFKLEDIEFYRQKYPDILIAVHPEC EPSVVSNADFSGSTSQIIEFVE KLSPNQKVAIGTESHLVNRLKAKRHHQ- 276 NADA_METTH DKTIIPIPEEGHCYVHKMFTAGDVMAAKEKYPEAELLIHPEC DPEVQELADHILSTGGMLRRVL ESDA-ESFIIGTEVDMTTRISLESD 248 NADA_METJA DKKVIAIPEGGGCYVHKKFTIDDLKRVKSKYPNAKVLIHPEC SPELQDNADYIASTSGILRIVL ESDD-EEFIIGTEVGMINRLEIELEKL- 250 NADA_PYRHO GKKIIPVPSKGHCYVHQKFTLDDVERAKKLHPNAKLMIHPEC IPEVQEKADIIASTGGMIKRAC EWD EWVVFTEREMVYRLRKLYPQ 244 NADA_AQUAE PEKEFIIWK-GFCPPHFEFTYKELEKLKEMYPDAKVAVHPEC HPRVIELADFVGSTSQILKYAT SVDA-KRVIVVTEVGLKYTLEKINPNKE 264 NADA_SYNY3 EMVLWQGSCIVHETFSERRLLELKTQYPQAEIIAHPEC EKAILRHADFIGSTTALLNYSG KSQG-KEFIVGTEPGIIHQMEKLSPSKQ 260 NADA_SYNEC EMVLWQGSCIVHETFSERRLLELKTQYPQAEIIAHPEC EKAILRHADFIGSTTALLNYSG KSQG-KEFIVGTEPGIIHQMEKLSPSKQ 260 NADA_CYAPA DLLLWPGSCIVHETFSEKKIFEFQSLYPTAEVIAHPEC EPTILKHANYIGSTTSLLQYVK NSKK-TTFIVITEPGIIHQMKKSCPEKQ 270 NADA_EHRCR KMILWPGTCIVHESFSERELIDMMVRHDKAYVLAHPEC PGHLLKYAHFIGSTTQLLKFSS DNPN-SEFIVLTEEGIIHQMKKVSSGST 253 NADA_MYCLE NVYVWMGECHVHAGINGDELVDQARANPDAELFVHPECGCSTSALYLAGEGAFPPDRVKILSTGGMLTAAR QTQY-RKILVATEVGMLYQLRRAAPEID 293 NADA_MYCTU NLHVWAGEC HVHAGINGDELADQARAHPDAELFVHPECGCATSALYLAGEGAFPAERVKILSTGGMLEAAH TTRA-RQVLVATEVGMLHQLRRAAPEVD 290 NADA_ATHAL HSLDSIKSLLPRLHYFQEGTCIVHHLFGHEVVERIKYMYCDAFLTAHLEVPG EMFSLAMEAKKREMGVVGSTQNILDFIKQKVQEAVDRNVDDHLQFVLGTESGMVTSIVAVIRSLL 355 NADA_OSATI HNKKSINALLPRLHYYQDGNCIVHDMFGHEVVDKIKEQYCDAFLTAHFEVPG EMFSLSMEAKTRGMGVVGSTQNILDFIKNHLMEALDRNIDDHLQFVLGTESGMITSIVAAVRELF 327 * * * : : : * * ** :: : *: : : NADA_ECOLI LLEAPTAGEG ATCRSCAHCPWMAMNGLQAIAEALEQEGSN HEVHVDERLRERALVPLN 336 NADA_SALTY LLEAPTAGEG ATCRSCAHCPWMAMNGLKAIAEGLEQGGAA HEIQVDAALREGALLPLN 336 NADA_BACSU QIESLN PDMCPCLTMNRIDLPHLLWSLEQIEKGEP SGVIKVPKAIQEDALLALN 362 NADA_HELPY NTFILS STLALCPTMNETTLKDLFEVLKAYKNHRA YNTIELKDEVARLAKLALT 330 NADA_METTH KKTIPL LEEAICENMKLHTLEKVKNSLINEEF VVTVPDEIARRARRAVE 297 NADA_METJA GKKKTLIPL RKDAICHEMKRITLEKIEKCLLEERY EIKLEKEIIEKAQKAIE 302 NADA_PYRHO KKFYPA REDAFCIGMKAITLKNIYESLKDMKY KVEVPEEIARKARKAIE 293 NADA_AQUAE YIFPQSMNY CGTVYCCTMKGITLPKVYETLKNEIN EVTLPKDIIERARRPIE 316 NADA_SYNY3 FIPLPNNSN CDCNECPYMRLNTLEKLYWAMQRRSP EITLPEATMAAALKPIQ 312 NADA_SYNEC FIPLPNNSN CDCNECPYMRLNTLEKLYWAMQRRSP EITLPEATMAAALKPIQ 312 NADA_CYAPA FLALPTVSG CACNECPHMRLNTLEKLYLAMKTRSP QIEIPESILLNAKKPIE 322 NADA_EHRCR FYIVKTSDSG G-CVSCSKCPHMRLNTLEKLYLCLKNGYP EITLDPEISSMAKRSLD 308 NADA_MYCLE FRAVNDRAS CKYMKMITPGALLRCLVEGTD EVHVDSEIAAAGRRSVQ 340 NADA_MYCTU FRAVNDRAS CKYMKMITPAALLRCLVEGAD EVHVDPGIAASGRRSVQ 337 NADA_ATHAL G SSANSKLKVEVVFPVSSDSMTKTSSDSSNSIKVGDVA LPVVPGVAGGEGCSIHGGCASCPYMKMNSLSSLLKVCHKLPDLENVYGGFIAERFKRQTPQGKLIADVGCEPIL 467 NADA_OSATI DSYKTSQQSANIEVEIVFPVSSDAVSNTSVNGSHHLDSSTVTDLDNVSVVPGVSSGEGCSIHGGCASCPYMKMNSLRSLLKVCHQLPDRDNRLVAYQASRFNAKTPLGKLVAEVGCEPIL 447 * * : . .: NADA_ECOLI RMLDFAATLRG 347 NADA_SALTY RMLDFAATLRA 347 NADA_BACSU RMLSIT 368 NADA_HELPY KMMELS 336 NADA_METTH RMIRVSE 304 NADA_METJA RMLRI 307 NADA_PYRHO RMLEMSK 300 NADA_AQUAE RMLELS 322 NADA_SYNY3 RMLAMS 318 NADA_SYNEC RMLAMS 318 NADA_CYAPA RMLEMSN 329 NADA_EHRCR AMLKMS 314 NADA_MYCLE RMIEIGLPGGGE 352 NADA_MYCTU RMIEIGHPGGGE 349 NADA_ATHAL HMRHFQANKELPDKLVHQVLSCESKR 493 NADA_OSATI HMRHFQATKRLPDKLVHHVIHGKGEPTS 475 * . NadA and NadB from B. subtilis I. Marinoni et al. 5096 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS a role for NadA in vivo, as its substitution with a serine conferred a clear Nic ) phenotype. Characterization of NadB NadB cloning, expression and purification were carried out as described in Experimental procedures. Typically, about 60 mg of pure enzyme was isolated from 6 L of bacterial growth medium. The homogeneity of the preparation was confirmed by SDS ⁄ PAGE and N-terminal sequence analysis (data not shown). The spectral properties of the flavoenzyme (Fig. 6) were very similar to those of NadB from E. coli, although the absorbance maximum was shifted to a Table 1. Identification of [Fe–S] cluster-binding residues of NadA. All of the cysteine residues were mutated to serine, and the mutants were probed for iron content and enzymatic activity in a glove box using fumarate as electron acceptor for NadB. A double mutant (C318S ⁄ C320S) was also obtained and tested. +, ability to grow in minimal medium without nicotinic acid; ), requirement for 0.5–50 mgÆmL )1 nicotinic acid for growth in minimal medium. ND, not determined. Enzyme Iron content ⁄ mol protein Enzymatic activity under anaerobic conditions (UÆmg )1 ) Nic phenotype of B. subtilis Wild-type 3.8 ± 0.3 0.050 ± 0.008 + C82S 4.3 ± 0.5 0.048 ± 0.010 + C110S 0.4 ± 0.1 No activity ) C230S 0.6 ± 0.2 No activity ) C259S 4.5 ± 0.5 0.038 ± 0.007 + C318S 3.3 ± 0.6 0.033 ± 0.005 ) C320S 1.5 ± 0.3 No activity ) C318S ⁄ C320S 0.3 ± 0.2 No activity ND Fig. 5. In vivo Nic phenotype verification of NadA mutants. Growth after 24 h at 37 °C of the wild-type (WT) (PB168, trpC 2 ) and mutated derivative B. subtilis strains, obtained by allelic switch. The strains were streaked on minimal Davis and Mingioli agar medium [43] in the presence of 0.5% glucose, 50 lgÆmL )1 tryptophan and, where indicated, of nicotinic acid (Nic, 50 lgÆmL )1 ). Fig. 4. Multiple alignment of NadA primary sequences from different bacteria and plants. Conserved cysteines are indicated in bold and labeled with arrows. All the cysteines of Bacillus subtilis, mutated to serine in the present work, are shaded in gray. The residues involved in the binding of malate in NadA from Pyrococcus horikoshii are indicated in bold. NADA_ECOLI: from Escherichia coli (Swiss Prot accession number P11458). NADA_SALTY: from Salmonella typhimurium (Swiss Prot accession number P24519). NADA_BACSU: from B. subtilis (Swiss Prot accession number O32063). NADA_HELPY: from Helicobacter pylori (Swiss Prot accession number O25910). NADA_METTH: from Methanobacterium thermoautotrophicum (Swiss Prot accession number O27855). NADA_METJA: from Methanococcus jannaschii (Swiss Prot accession number Q57850). NADA_PYRHO: from P. horikoshii (Swiss Prot accession number O57767). NADA_AQUAE: from Aquifex aeolicus (Swiss Prot accession number O67730). NADA_SYNY3: from Synechocystis sp. strain PCC 6803 (Swiss Prot accession number P74578). NADA_SYNEC: from Synechocystis (GenBank accession number NP_442873). NADA_CYAPA: from Cyanophora paradoxa (Swiss Prot accession number P31179). NADA_EHRCR: from Ehrlichia chaffeensis (Swiss Prot accession number O05104). NADA_MYCLE: from Mycobacterium leprae (Swiss Prot accession number Q49622). NADA_MYCTU: from Mycobacterium tuberculosis (Swiss Prot acces- sion number O06596). NADA_ATHAL: from Arabidopsis thaliana (GenBank accession number NP_199832). NADA_OSATI: from Oryza sativa (GenBank accession number ABA_97161). I. Marinoni et al. NadA and NadB from B. subtilis FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5097 lower wavelength (444 nm instead of 452 nm). A rec- onstitutable apoprotein was obtained and was utilized to determine the dissociation constant for the FAD– enzyme complex by the ultrafiltration method, both in the presence and in the absence of 10 mm fuma- rate [9], giving values of 4.46 ± 0.5 lm for the free enzyme and 1.6 ± 0.5 lm for the complex with fumarate. These results were very similar to the data described for the enzyme from E. coli (3.8 lm in the presence of fumarate and 0.6 lm in the absence of fumarate, respectively [14]), suggesting that, as in the enzyme from E. coli, in NadB from B. subtilis the presence of the substrate fumarate in the incubation mixture does stabilize the holoenzyme significantly. In contrast, the dissociation constant for the FAD– enzyme complex did not change if the apoenzyme was incubated with the coenzyme in the presence of a stoichiometric amount of NadA, suggesting that this protein does not influence the binding of FAD to NadB. The enzyme from B. subtilis showed typical flavin fluorescence, with excitation and emission maxima at 450 nm and 520 nm, respectively. The binding of FAD did not quench the protein fluorescence (excitation at 295 nm, emission at 340 nm) (data not shown). More- over, upon addition of NadA in NadA ⁄ NadB ratios of 1 : 1 and 2 : 1, the flavin coenzyme fluorescence prop- erties were still the same as in the absence of NadA. The aggregation state of pure NadB was determined by gel filtration. In accordance with the results for the enzyme from E. coli [9], NadB from B. subtilis is a dimer of 115 kDa in the absence of NaCl and a mono- mer of 55 kDa in the presence of 150 mm NaCl. After incubation with pure NadA in ratios of 1 : 1 or 2 : 1, under either aerobic or anaerobic conditions, gel filtra- tion experiments did not show any peaks with a molec- ular weight equal to the sum of NadA and NadB, suggesting that the two proteins do not form a stable complex in such conditions. The binding of dicarboxylic compounds caused spec- tral changes similar to those observed in NadB from E. coli, as shown in Fig. 6A for the binding of fuma- rate. However, the corresponding dissociation con- stants were higher for the enzyme from B. subtilis,as shown in Table 2, which reports the values calculated for the enzyme from B. subtilis, as well as the corre- sponding K d measured for NadB from E. coli in 50 mm potassium phosphate buffer (pH 8.0) and 20% glycerol. The opposite was observed for the product iminoaspartate, which bound more tightly to the enzyme from B. subtilis (Table 2). In keeping with this observation, the enzyme showed pronounced product inhibition when the l-aspartate oxidase activity was checked at 0.24 mm oxygen and the l-aspartate–fuma- rate oxidoreductase activity was determined under anaerobic conditions. NadB shows three enzymatic activities: l-aspartate– oxygen oxidoreductase activity, fumarate reductase activity, and l-aspartate–fumarate oxidoreductase activity. Regarding the l-aspartate oxidase activity, using oxygen as electron acceptor, the apparent K m (1.0 ± 0.6 mm) and the k cat (10.8 ± 1.0 min )1 ) were calculated, and are reported in Table 3. NadB from B. subtilis could use fumarate as electron acceptor with a k cat ⁄ K m ratio comparable to the one reported for the enzyme from E. coli. As far as the l-aspartate–fuma- rate oxidoreductase activity was concerned, the double substrate inhibition pattern described for NadB from E. coli [14] was also present in the protein from B. sub- tilis, but in the latter case the substrate inhibition was greater and the turnover number and the other kinetic Fig. 6. Purification and spectral properties of NadB from Bacil- lus subtilis: the visible absorption spectrum of NadB in 50 m M potassium phosphate buffer (pH 8.0) containing 20% glycerol, at 25 °C, before ( _______ ) and after (- - -) addition of 20 mM fumarate. Inset: Benesi–Hildebrand plot for the binding of fumarate. K d = 4.4 mM. Table 2. Dissociation constants for the binding of dicarboxylic com- pounds to NadB from Bacillus subtilis. The dissociation constants for dicarboxylic ligands were measured spectrophotometrically by addition of small volumes of concentrated stock solutions to sam- ples containing about 10–25 l M holoenzyme at 25 °Cin50mM potassium phosphate buffer (pH 8.0) and 20% glycerol. Iminoaspar- tate was produced by an enzymatic system consisting of D-aspar- tate ⁄ D-aspartate oxidase to produce iminoaspartate in situ free of excess reagents, using a concentration of D-aspartate of 300 lM. The corresponding values for the enzyme from Escherichia coli were evaluated under the same conditions for comparison. Ligands B. subtilis NadB E. coli NadB Fumarate 4.4 ± 0.5 m M 1.14 ± 0.50 mM Succinate 55 ± 3 mM 2.7 ± 0.7 mM D -Aspartate 32 ± 2 mM 9±1mM Oxaloacetic acid 0.5 ± 0.3 mM 1.7 ± 0.2 mM Iminoaspartate 0.32 ± 0.10 lM 1.0 ± 0.5 lM NadA and NadB from B. subtilis I. Marinoni et al. 5098 FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS parameters could not be accurately determined, as it was impossible to work with high l-aspartate or fuma- rate concentrations. However, taken together, the data suggest that in the case of NadB from B. subtilis, fumarate can replace oxygen as electron acceptor, simi- larly to what has been described for the enzyme from E. coli, and that the two proteins present very similar biochemical properties. NadA–NadB interaction As reported above, pure and active NadA and NadB from B. subtilis were obtained in solution in reasonable amounts, opening the possibility for an investigation of the complex between NadA and NadB. Such a multienzymatic complex (sometimes referred to as the ‘quinolinate synthase complex’) has never been observed, but has been proposed on the grounds of the following indirect observations: (a) iminoaspartate, the product of NadB and substrate of NadA, is unsta- ble in solution, and consequently it is unlikely that it has to reach NadA simply by diffusing through the cell; and (b) partial copurification of the two wild-type enzymes has been obtained in E. coli [34]. On the other hand, it has been reported that NadA from E. coli can form quinolinate using iminoaspartate produced by d-aspartate oxidase [2] or chemically generated in the assay mixture [35]. Moreover, in T. maritima, NadB is substituted by an NADP-dependent l-aspartate dehy- drogenase to produce iminoaspartate [17]. In an attempt to solve this issue, we utilized the following different approaches. The existence of species-specific interactions between NadA and NadB in quinolinate formation was investi- gated by evaluating the enzymatic activity of NadA from B. subtilis in the presence of 20 lg of NadB from E. coli, using either fumarate or oxygen as electron acceptor for NadB. The specific activity was 0.04 ± 0.02 lmolÆmin )1 mg )1 in the presence of fuma- rate and 0.027 ± 0.01 lmolÆmin )1 Æmg )1 using oxygen. If compared with the results obtained using NadB from B. subtilis reported above, these data suggest that NadA is unable to discriminate between NadB from B. subtilis and from E. coli, and that the interaction between the two proteins is not species-specific in this case. As the presence of His-tags or GST-tags does not affect the activity and properties of NadA, it was pos- sible to apply an affinity capture protocol using recom- binant GST–NadA or NadA–His. NadA fused to GST and bound to glutathione–Sepharose (1 mL of resin saturated with NadA) was incubated in batches with: (a) pure NadB from B. subtilis (NadA ⁄ NadB ratio 1 : 1 or 2 : 1); (b) a homogenate obtained from E. coli cells overexpressing NadB from B. subtilis; or (c) a homogenate of B. subtilis cells (500 lg of total proteins). The incubation took place under anaerobic conditions at room temperature for up to 30 min in: (a) 50 mm Tris ⁄ HCl (pH 7.5) and 0.15 m NaCl; or (b) 50 mm Tris ⁄ HCl (pH 8.0) and 10 mm b-mercaptoetha- nol. A control experiment was set up by incubating pure NadB with glutathione–Sepharose without NadA in order to rule out the possibility of unspecific binding of NadB to the resin. After extensive washing, the pro- teins were eluted and subjected to 11% SDS ⁄ PAGE. The gels were either stained by silver or Coomassie blue or electroblotted onto a poly(vinylidene difluo- ride) membrane for N-terminal sequence analysis. A band corresponding to NadB could be detected in the samples obtained from the incubation with pure NadB and with the homogenate of E. coli overexpressing NadB from B. subtilis. Moreover, the comparison car- ried out with the control showed that this band was not due to unspecific binding of NadB to the resin. The same data were obtained if pure NadB was incub- ated with NadA–His bound to an Ni 2+ –nitrilotriacetic acid resin, suggesting that the binding is not dependent on the presence of a tag either at the N-terminus or at Table 3. Kinetic parameters for the three activities of NadB. The activity assays were carried out as described in Experimental procedures. The corresponding values for the enzyme from Escherichia coli are reported in Tedeschi et al. [9,14]. Activity NadB from B. subtilis NadB from E. coli K m L –Asp (mM) K m fumarate (mM) k cat (s )1 ) k cat ⁄ K m L -Asp (s )1 M )1 ) k cat ⁄ K m fumarate (s )1 M )1 ) K m L –Asp (mM) K m fumarate (mM) k cat (s )1 ) k cat ⁄ Km L -Asp (s )1 M )1 ) k cat ⁄ Km fumarate (s )1 M )1 ) L-Aspartate–oxygen oxidoreductase 1.0 ± 0.5 0.18 ± 0.02 180 1.74 0.465 267.23 Fumarate reductase 1.6 ± 0.6 15.4 ± 0.8 9625 0.048 0.27 5625 L-Aspartate–fumarate oxidoreductase 20.0 ± 3 1.43 ± 0.9 0.50 ± 0.3 25 350 2.70 2.50 5.55 2055.55 2220 I. Marinoni et al. NadA and NadB from B. subtilis FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5099 [...]... Tecnologica, Italy) and FIRST 2007 (University of Milano, Italy), FAR 2005 and FAR 2006 (University of Pavia, Italy) We thank E Andreoli for expert and careful technical support, N Marchesi and F Rocchi for help in the construction of some of the B subtilis nadA NadA and NadB from B subtilis mutants, and F Corniola for assistance in figure preparation References 1 Berger F, Ramirez-Hernandez MH & Ziegler... gene and part of the 3¢-end of the nadC coding sequence and the promoter and 5¢-end of the safA coding sequence, was PCRamplified from the B subtilis 168 (trpC2) wild-type strain chromosome, using the primers SSafA and N1911 listed in 5102 Table S1, and cloned in the vector pMAD [33], yielding the vector pMADNAD11 For the construction of the pMADDNadA vector for allelic replacement, we cloned in tandem... of FEBS Journal 275 (2008) 5090–5107 ª 2008 The Authors Journal compilation ª 2008 FEBS 5105 NadA and NadB from B subtilis 12 13 14 15 16 17 18 19 20 21 22 23 I Marinoni et al l-aspartate oxidase, the first enzyme in the bacterial de novo biosynthesis of NAD Acta Crystallogr 55, 549–551 Tedeschi G, Negri A, Ceciliani F, Mattevi A & Ronchi S (1999) Structural characterization of l-aspartate oxidase and. .. Simonic T, Faotto L & Ronchi S (1996) l-aspartate oxidase from Escherichia coli II Interaction with C4-dicarboxylic acids and identification of a novel l-aspartate: fumarate oxidoreductase activity Eur J Biochem 239, 427–433 10 Tedeschi G, Zetta L, Negri A, Mortarino M, Ceciliani F & Ronchi S (1997) Redox potentials and quinone reductase activity of l-aspartate oxidase from E coli Biochemistry 36, 16221–16230... oxidase to produce iminoaspartate in situ free of excess reagents, using a concentration of d-aspartate (300 lm) that was low enough to avoid the formation of the complex with NadB The amount of NadB–iminoaspartate complex can be directly estimated from the increase in absorbance at 495 nm The koff of iminoaspartate from NadB was measured as described in Mortarino et al [8] In detail, NadB from B subtilis. .. acid-labile sulfide and sulfhydryl groups Methods Enzymol 53, 275–277 41 Yamada R, Nagasaki H, Wabayashi Y & Iwashima A (1988) Presence of d-aspartate oxidase in rat liver and mouse tissues Biochim Biophys Acta 965, 202–205 NadA and NadB from B subtilis 42 Negri A, Tedeschi G, Ceciliani F & Ronchi S (1999) Purification of beef kidney d-aspartate oxidase overexpressed in E coli and characterizaztion of its redox... catalyzed by the NadA and NadB proteins, which belong to a large family of proteins with homologs in bacteria, archaeans and plants Despite the important role played by these enzymes in NAD metabolism in bacteria, NadB has been characterized only from E coli and S tokodaii, and the biochemical and structural properties of NadA are still largely unknown The experimental findings on NadB from B subtilis reported... phosphate buffer (pH 8) and 20% glycerol containing 10 mm oxaloacetate and 0.3 m ammonium sulfate to produce iminoaspartate Following removal of excess reagents by gel filtration on a PD10 column, the release of iminoaspartate from the complex with NadB was followed at 495 nm both in the presence and in the absence of a stoichiometric amount of NadA checked by using 10 mm succinate instead of l-aspartate in... dissociation of oxaloacetate, but not of iminoaspartate The iminoaspartate itself was stabilized upon binding to NadB, as the observed rate constant of hydrolysis of free iminoaspartate to oxaloacetate and Fig 7 NadA–NadB interaction Decay of the NadB–iminoaspartate complex in the presence of NadA with an NadA ⁄ NadB ratio of 1 : 1, under strictly anaerobic conditions The visible absorption spectrum of the... and NadB from B subtilis I Marinoni et al the C-terminus of NadA (data not shown) Changing the incubation buffer, the condition with regard to aerobic or anaerobic status, incubation temperature and time as well as adding 10 mm l-aspartate or fumarate to the incubation mixture did not affect the results On the other hand, we were unable to detect any binding upon incubation with a homogenate of B subtilis . Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis Ilaria Marinoni 1 , Simona Nonnis 2 , Carmine Monteferrante 1 ,. proteins from B. subtilis and E. coli. Results and Discussion NadA cloning and protein purification and characterization In order to optimize the heterologous production and purification of B. subtilis. contained 70 lg of NadA, 30 lgof NadB and 1 mm fumarate under anaerobic conditions. AB Fig. 1. Production and purification of NadA from Bacillus subtilis. (A) 11% SDS ⁄ PAGE of NadA–His before and after