Báo cáo khoa học: Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis potx

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-aspartat

Characterization of -aspartate oxidase and quinolinate synthase from Bacillus subtilis

Ilaria Marinoni1, Simona Nonnis2, Carmine Monteferrante1, Peter Heathcote3, Elisabeth Ha¨rtig4, Lars H Bo¨ttger5, Alfred X Trautwein5, Armando Negri2, Alessandra M Albertini1and

Gabriella Tedeschi2

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 subtilispossesses 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.

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.

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 interacinforma-tion

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

His6-tag at the N-terminus, NadA with a His6-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

1 L 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 lg of NadB and 1 mm fumarate under anaerobic conditions

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 (- - -).

An apparent Kmof 0.36 ± 0.05 mm was calculated for

DHAP at 25C, using oxygen as electron acceptor

The specific activity of NadA from B subtilis was

0.05 ± 0.01 lmolÆmin)1Æmg)1 in the presence of

fuma-rate as electron acceptor for NadB, and 0.027 ±

0.01 lmolÆmin)1Æmg)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)1Æmg)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)1Æmg)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 DEQ= 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,

DEQ= 0.53 mmÆs)1 and G = 0.35 mmÆs)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).

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 Fe2.5+ 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 Fe2.5+ 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; dI= 0.42 mmÆs)1, DEQI= 1.05 mmÆs)1,

GI= 0.43 mmÆs)1) represents tetrahedral-sulfur-coordi-nated Fe2.5+sites, and doublet II (dotted line; the rela-tive area 25% was fixed in the fit; dII= 0.52 mmÆs)1,

DEQII = 1.09 mmÆs)1, GII= 0.42 mmÆs)1) represents the tetrahedral-coordinated Fe2.5+ site with the cyste-ine ligand replaced by a noncystecyste-ine 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 Cu2+, 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 g1= 2.054 and

g2,3= 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 pKa 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.

activity [20,21], but no information is available

regard-ing the residues involved in bindregard-ing 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

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

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_METTH -MLNQLQRDILRLKKEKNAIILAHNYQSREIQEIADFKG -DSLELCIEASRIEG KDIVVFCGVDFMAETAYILNPDK 75

NADA_PYRHO -MDLVEEILRLKEERNAIILAHNYQLPEVQDIADFIG -DSLELARRATRVD -ADVIVFAGVDFMAETAKILNPDK 72

NADA_SYNY3 -MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG -DSLGLSQQAASTD -ADVIVFAGVHFMAETAKILNPHK 85

NADA_SYNEC -MFTAVAPPQETLP RDLVGAIQSLKKELNAVILAHYYQEAAIQDIADYLG -DSLGLSQQAASTD -ADVIVFAGVHFMAETAKILNPHK 85

NADA_EHRCR -MKELDVIT -LLQEIRHLAQESNAVILAHYYQDSEIQDIADFIG -DSLELSRKAATTT -ADVIVFCGVYFMAEVAKIINPAK 78

NADA_MYCLE MTVLNGMEPLAGDMTVVIAGITDSPVGYAGVDGDEQWATEIRRLTRLRGATVLAHNYQLPAIQDIADYVG -DSLALSRIAAEVP -EETIVFCGVHFMAETAKILSPNK 106

NADA_ATHAL VPSFEPFPSLVLTAHGIEAKGSFAQAQAKYLFPEESRVEELVNVLKEKKIGVVAHFYMDPEVQGVLTAAQKHWPHISISDSLVMADSAVTMAKAGCQFITVLGVDFMSENVRAILDQAGF 120 NADA_OSATI -MFLSPNESKTSELVKSLREKKIGIVAHFYMDPEVQGILTASKKHWPHIHISDSLVMADSAVKMAEAGCEYITVLGVDFMSENVRAILDQAGY 92 : * * : *** : : ** ** * :

NADA_ECOLI -TILMPT-LQAECSLDLGCPVEEFNAFCDAHPDRT -VVVYANTSAAVKARAD -WVVTSSIAVELIDHL DSLGEKIIWAPDKHLGRYVQKQTGG - 191

NADA_SALTY -TILMPT-LAAECSLDLGCPIDEFSAFCDAHPDRT -VVVYANTSAAVKARAD -WVVTSSIAVELIEHL DSLGEKIIWAPDRHLGNYVQKQTGA - 191

NADA_HELPY -QVIMP KLSCCSMARMIDSHYYDRSVHLLKECGVKEFYPITYINSNAEVKAKVAKDD-GVVCTSRNASKIFNHA LKQNKKIFFLPDKCLGENLALENGLKSAILGANS - 182

NADA_METJA -KVLMPEIEGTQCPMAHQLPPEIIKKYRELYPEAP -LVVYVNTTAETKALAD -ITCTSANADRVVNS LDADTVLFGPDNNLAYYVQKRT - 160

NADA_AQUAE -KVLHPN-PESGCPMADMITAKQVRELREKHPDAE -FVAYINTTADVKAEVD -ICVTSANAPKIIKK LEAKKIVFLPDQALGNWVAKQV - 174

NADA_SYNEC -LVLLPD-LEAGCSLADSCPPREFAEFKQRHPDHL -VISYINCTAEIKALSD -IICTSSNAVKIVQQ -LPPDQKIIFAPDRNLGRYVMEQTGR - 173

NADA_EHRCR -KVLLPD-LNAGCSLADSCDAESFKKFRELHKDCV -SITYINSLAEVKAYSD -IICTSSSAEKIIRQ -IPEEKQILFAPDKFLGAFLEKKTNR - 166

NADA_MYCTU -TVLIPD-QRAGCSLADSITPDELRAWKDEHPGAV -VVSYVNTTAAVKALTD -ICCTSSNAVDVVAS -IDPDREVLFCPDQFLGAHVRRVTGRK - 192

NADA_OSATI SKVGVYRMSSDQIGCSLADAASSSAYTHFLKEASRSPPS LHVIYINTSLETKAHAHELVPTITCTSSNVVATILQAFAQIPGLNVWYGPDSYMGANIADLFQRMAVMSDEEIAEVHPS 210 *.: : * * ** ** : : ** :

NADA_ECOLI -DILCWQGACIVHDEFKTQALTRLQEEYPDAAILVHPES -PQAIVDMADAVGSTSQLIAAAK -TLPH-QRLIVATDRGIFYKMQQAVPDKE 278 NADA_BACSU V -AESGHTNVKVILWKGHCSVHEKFTTKNIHDMRERDPDIQIIVHPEC -SHEVVTLSDDNGSTKYIIDTIN -QAPAGSKWAIGTEMNLVQRIIHEHPDK- 308 NADA_METTH -DKTIIPIPEEGHCYVHKMFTAGDVMAAKEKYPEAELLIHPEC -DPEVQELADHILSTGGMLRRVL -ESDA-ESFIIGTEVDMTTRISLESD - 248

NADA_PYRHO -GKKIIPVPSKGHCYVHQKFTLDDVERAKKLHPNAKLMIHPEC -IPEVQEKADIIASTGGMIKRAC -EWD -EWVVFTEREMVYRLRKLYPQ 244

NADA_AQUAE -PEKEFIIWK-GFCPPHFEFTYKELEKLKEMYPDAKVAVHPEC -HPRVIELADFVGSTSQILKYAT -SVDA-KRVIVVTEVGLKYTLEKINPNKE 264 NADA_SYNEC -EMVLWQGSCIVHETFSERRLLELKTQYPQAEIIAHPEC -EKAILRHADFIGSTTALLNYSG -KSQG-KEFIVGTEPGIIHQMEKLSPSKQ 260 NADA_EHRCR -KMILWPGTCIVHESFSERELIDMMVRHDKAYVLAHPEC -PGHLLKYAHFIGSTTQLLKFSS -DNPN-SEFIVLTEEGIIHQMKKVSSGST 253 NADA_MYCTU -NLHVWAGECHVHAGINGDELADQARAHPDAELFVHPECGCATSALYLAGEGAFPAERVKILSTGGMLEAAH -TTRA-RQVLVATEVGMLHQLRRAAPEVD 290 NADA_OSATI HNKKSINALLPRLHYYQDGNCIVHDMFGHEVVDKIKEQYCDAFLTAHFEVPG -EMFSLSMEAKTRGMGVVGSTQNILDFIKNHLMEALDRNIDDHLQFVLGTESGMITSIVAAVRELF 327 * * * : : : * * ** :: : *: : :

NADA_ECOLI LLEAPTAGEG -ATCRSCAHCPWMAMNGLQAIAEALEQEGSN -HEVHVDERLRERALVPLN 336 NADA_SALTY LLEAPTAGEG -ATCRSCAHCPWMAMNGLKAIAEGLEQGGAA -HEIQVDAALREGALLPLN 336 NADA_HELPY -NTFILS -STLALCPTMNETTLKDLFEVLKAYKNHRA -YNTIELKDEVARLAKLALT 330 NADA_METJA GKKKTLIPL -RKDAICHEMKRITLEKIEKCLLEERY -EIKLEKEIIEKAQKAIE 302 NADA_AQUAE YIFPQSMNY -CGTVYCCTMKGITLPKVYETLKNEIN -EVTLPKDIIERARRPIE 316 NADA_SYNEC FIPLPNNSN -CDCNECPYMRLNTLEKLYWAMQRRSP -EITLPEATMAAALKPIQ 312 NADA_EHRCR FYIVKTSDSG -G-CVSCSKCPHMRLNTLEKLYLCLKNGYP -EITLDPEISSMAKRSLD 308 NADA_MYCTU FRAVNDRAS -CKYMKMITPAALLRCLVEGAD -EVHVDPGIAASGRRSVQ 337 NADA_OSATI DSYKTSQQSANIEVEIVFPVSSDAVSNTSVNGSHHLDSSTVTDLDNVSVVPGVSSGEGCSIHGGCASCPYMKMNSLRSLLKVCHQLPDRDNRLVAYQASRFNAKTPLGKLVAEVGCEPIL 447 * * : .:

NADA_ECOLI RMLDFAATLRG - 347

NADA_BACSU RMLSIT - 368

NADA_METTH RMIRVSE - 304

NADA_METJA RMLRI - 307

NADA_AQUAE RMLELS - 322

NADA_SYNEC RMLAMS - 318

NADA_EHRCR AMLKMS - 314

NADA_MYCTU RMIEIGHPGGGE - 349

NADA_OSATI HMRHFQATKRLPDKLVHHVIHGKGEPTS 475 *

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)1nicotinic 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

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)1tryptophan 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 accesacces-sion number NP_199832) NADA_OSATI: from Oryza sativa (GenBank accession number ABA_97161).

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 Kd 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 Km

(1.0 ± 0.6 mm) and the kcat (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 kcat⁄ Kmratio 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 m M fumarate.

Inset: Benesi–Hildebrand plot for the binding of fumarate.

K d = 4.4 m M

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 C in 50 m M 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 l M The corresponding values for the enzyme from Escherichia coli were evaluated under the same conditions for comparison.

Oxaloacetic acid 0.5 ± 0.3 m M 1.7 ± 0.2 m M Iminoaspartate 0.32 ± 0.10 l M 1.0 ± 0.5 l M

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)1mg)1in the presence of

fuma-rate and 0.027 ± 0.01 lmolÆmin)1Æmg)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 Ni2+–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

K m L –Asp

(m M )

K m fumarate

(m M ) kcat(s)1)

kcat⁄

K m L -Asp

(s)1M )1)

kcat⁄

K m fumarate

(s)1M )1)

K m L –Asp

(m M )

K m fumarate

(m M )

k cat

(s)1)

kcat⁄

Km L -Asp

(s)1M )1)

kcat⁄

Km fumarate

(s)1M )1)

L -Aspartate–oxygen

oxidoreductase

L -Aspartate–fumarate

oxidoreductase