Báo cáo khoa học: Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli pdf

In this report, we examined the in vivo and in vitro incorporation of metal ions of molybdenum, tungsten, vanadium, copper, cadmium and arsenic into the MPT cofactor.. Our results sugges

binding to the dithiolene moiety of molybdopterin in

Escherichia coli

Meina Neumann and Silke Leimku¨hler

Department of Proteinanalytics, Institute of Biochemistry and Biology, University of Potsdam, Germany

Molybdenum is the only second-row transition metal

that is required by most living organisms, and the few

species that do not require molybdenum use tungsten,

which lies immediately below molybdenum in the

Peri-odic Table [1,2] Molybdenum- and

tungsten-contain-ing enzymes often catalyse the same types of reaction;

while molybdenum-containing enzymes are found in

all aerobic organisms, including humans,

tungsten-containing enzymes are found only in obligate,

typi-cally thermophilic, anaerobic bacteria and archaea

[3,4] The molybdenum cofactor (Moco) is the essential component of a group of redox enzymes which cata-lyse a variety of transformations at carbon, sulfur and nitrogen atoms More than 40 molybdenum and tung-sten enzymes have been identified in bacteria, archaea, plants and animals to date [5,6] Some of the better known Moco-containing enzymes include sulfite oxi-dase, xanthine dehydrogenase and aldehyde oxi-dase in humans [6], assimilatory nitrate reductase in plants [7] and dissimilatory nitrate reductase,

Keywords

copper; dithiolene; metal toxicity; Moco;

molybdenum

Correspondence

S Leimku¨hler, Institute of Biochemistry and

Biology, University of Potsdam, D-14476

Potsdam, Germany

Fax: +49 331 977 5128

Tel: +49 331 977 5603

E-mail: sleim@uni-potsdam.de

(Received 15 July 2008, revised

3 September 2008, accepted

19 September 2008)

doi:10.1111/j.1742-4658.2008.06694.x

Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain

of molybdopterin is a highly specific process that is catalysed by the MoeA and MogA proteins in Escherichia coli Ligation of molybdate to molybdopterin generates the molybdenum cofactor, which can be inserted directly into molybdoenzymes binding the molybdopterin form of the molybdenum cofactor, or is further modified in bacteria to form the dinu-cleotide form of the molybdenum cofactor The ability of various metals to bind tightly to sulfur-rich sites raised the question of whether other metal ions could be inserted in place of molybdenum at the dithiolene moiety of molybdopterin in molybdoenzymes We used the heterologous expression systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulf-oxide reductase in E coli to study the incorporation of different metal ions into the molybdopterin site of these enzymes From the added metal-con-taining compounds Na2MoO4, Na2WO4, NaVO3, Cu(NO3)2, CdSO4 and NaAsO2 during the growth of E coli, only molybdate and tungstate were specifically inserted into sulfite oxidase and dimethylsulfoxide reductase Other metals, such as copper, cadmium and arsenite, were nonspecifically inserted into sulfite oxidase, but not into dimethylsulfoxide reductase We showed that metal insertion into molybdopterin occurs beyond the step of molybdopterin synthase and is independent of MoeA and MogA proteins Our study shows that the activity of molybdoenzymes, such as sulfite oxidase, is inhibited by high concentrations of heavy metals in the cell, which will help to further the understanding of metal toxicity in E coli

Abbreviations

FeVco, iron–vanadium cofactor; hSO, human sulfite oxidase; hSO-MD, human sulfite oxidase Moco domain; ICP-OES, inductively coupled plasma-optical emission spectrometry; MGD, molybdopterin guanine dinucleotide cofactor; Moco, molybdenum cofactor; MPT,

molybdopterin; Wco, tungsten cofactor.

dimethylsulfoxide reductase and formate

dehydroge-nase in bacteria [8–10]

Moco biosynthesis has been studied extensively in

Escherichia coli using a combination of biochemical,

genetic and structural approaches [11,12], and

addi-tional insights have been provided by studies in

eukaryotes [13] The biosynthesis of Moco can be

divided into three general steps in all organisms: (a)

formation of precursor Z from GTP; (b) formation of

molybdopterin (MPT) by insertion of two sulfur atoms

into precursor Z; (c) insertion of molybdenum into the

dithiolene group of MPT, thus forming Moco In

bac-teria, such as E coli, an additional modification of

Moco occurs with the attachment of GMP to the

phosphate group of Moco, forming the MPT guanine

dinucleotide cofactor (MGD) [14,15] In total, more

than 10 genes are involved in the biosynthesis of Moco

in E coli, and highly conserved proteins have been

identified in other organisms

Molybdenum enters the cell as the soluble oxyanion

molybdate, for which high-affinity molybdate

trans-porters have been described in bacteria [11,16] and in

higher eukaryotes [17,18] In the step of molybdenum

insertion into MPT, the gene products of moeA and

mogAare involved in E coli It has been observed that

MoeA mediates molybdenum ligation to newly

synthe-sized MPT at low concentrations of molybdate, and

MogA helps to facilitate this step in vivo in an

ATP-dependent manner via an MPT-adenylate intermediate

[19,20] The crystal structure of the Arabidopsis

thali-ana Cnx1 G protein, a homologue of E coli MogA,

shows that copper is bound to the MPT dithiolene

sulfurs of the MPT–AMP complex [21] It has been

proposed that copper binding to MPT–AMP on Cnx1

is physiologically relevant and that, in vivo, copper

may serve to protect the dithiolene moiety prior to the

binding of molybdenum However, as several metal

ions are known to bind tightly to sulfur-rich sites, a

more recent report by Morrison et al [22] investigated

the effect of copper-limiting reaction conditions on

molybdoenzymes in E coli and Rhodobacter

sphaero-ides Their results demonstrated that the activities of

dimethylsulfoxide reductase and nitrate reductase were

not repressed under copper starvation, showing that

copper is not strictly required for the biosynthesis of

Moco in bacteria [22]

In addition to copper, various metals are known to

bind tightly to sulfur-rich sites, leading to the question

of whether other metal ions can bind to the dithiolene

moiety of Moco and be inserted into molybdoenzymes

Some of the most common environmental toxins are

cadmium and arsenic Arsenic is ubiquitous in the

environment and is most commonly found in an

insol-uble form associated with rocks and minerals [23,24]

In soluble form, arsenic occurs as trivalent arsenite [As(III)] and pentavalent arsenate [As(V)] Arsenate, a phosphate analogue, can enter cells via the phosphate transport system, and is toxic because it can interfere with normal phosphorylation processes by replacing phosphate [25] The competitive substitution of arse-nate for phosphate can lead to rapid hydrolysis of the high-energy bonds in compounds such as ATP Arse-nite has recently been demonstrated to enter cells at neutral pH by aqua-glyceroporins (glycerol transport proteins) in bacteria, yeast and mammals [26], and its toxicity lies in its ability to bind sulfhydryl groups of cysteine residues in proteins, thereby inactivating them Arsenite is considered to be more toxic than arsenate and can be oxidized to arsenate chemically or microbi-ally [27,28]

In contrast, cadmium is soluble as its bivalent cation

Cd2+, and Cd2+ions are readily taken up by bacterial and eukaryotic cells, presumably by the Mn2+uptake system [29] Cadmium toxicity may be caused by bind-ing to zinc bindbind-ing proteins, e.g proteins that contain zinc finger protein structures [30] Zinc and cadmium are in the same group in the Periodic Table, contain the same common oxidation state (+2) and, when ionized, are almost the same size As a result of these similarities, cadmium can replace zinc in many biologi-cal systems, in particular systems that contain sulfur ligands [31] Cadmium can be bound up to 10 times more strongly than zinc to certain biological systems, and is thus difficult to remove [31] In addition, cad-mium can replace magnesium and calcium in certain proteins [30]

Vanadium is chemically similar to molybdenum, and can replace molybdenum in its role in nitrogenase, form-ing the iron–vanadium cofactor (FeVco) [32] Like molybdenum, vanadium is available in anionic and cat-ionic forms, the most common being, under physiologi-cal conditions, vanadate (H2VO4)) and vanadyl (VO2+) [33] Vanadate can act as a competitor to phosphate (HPO4 )), or as a transition metal ion that competes with other metal ions in coordination with biogenic compounds Because of the low molecular weight of

VO3), like phosphate, the VO3)ion is able to permeate plasma membranes and the intestinal wall in humans with relative ease [34–36] Vanadate ions also mimic most of the rapid actions of insulin in the cell [37]

In this report, we examined the in vivo and in vitro incorporation of metal ions of molybdenum, tungsten, vanadium, copper, cadmium and arsenic into the MPT cofactor For in vivo studies, we heterologously pro-duced human sulfite oxidase (hSO) and R sphaeroides dimethylsulfoxide reductase in the presence of metal

ions in E coli and studied the incorporation of

metal-bound MPT into these enzymes Our results

demonstrate that only molybdate and tungstate are

specifically inserted into MPT and MGD in E coli

Bivalent copper and cadmium ions and trivalent

arse-nite can be inserted nonspecifically into MPT, which is

inserted into hSO, thus inhibiting enzyme activity

However, copper, cadmium and arsenite are not

inserted into bis-MGD containing dimethylsulfoxide

reductase in E coli Our results suggest that enzymes

containing the MPT form of Moco can easily be

inhib-ited by copper, cadmium, arsenic and other metal ions

binding to sulfur-rich sites, whereas

bis-MGD-contain-ing enzymes are rather protected from nonspecific

metal insertion

Results

Investigation of metal ion insertion into hSO

during heterologous expression in E coli

The expression of hSO in an E coli modC) strain [38]

results in an MPT-containing form of hSO, which is

free of metal ions at the MPT site (data not shown) It

has been shown previously that the addition of 100 lm

molybdate is sufficient to complement the modC)

phe-notype [39,40] Thus, the production of hSO in the

E coli modC) strain is an ideal system to study the

nonspecific insertion of metal ions, other than

molyb-date, into hSO In addition, both the E coli moeA)

and mogA) strains [38,41] were used for comparative

studies, as MoeA and MogA have been shown to be

involved in the specific insertion of molybdate into

MPT [19,20] Comparative studies of the metal

contents of hSO after production in E coli modC),

mogA) and moeA) strains should make it possible to

distinguish whether the metal ions are inserted

specifi-cally or nonspecifispecifi-cally into hSO For metal insertion

into hSO, the protein was produced in the E coli

modC), moeA) and mogA) strains in the presence of

100 lm of Na2MoO4, Na2WO4, NaVO3, Cu(NO3)2,

CdSO4or NaAsO2 For metal analysis, hSO was

puri-fied after expression In addition, the uptake of metal

ions was analysed in E coli cell extracts As shown in

Fig 1A, the addition of 100 lm molybdate during

expression in the modC) strain resulted in a 65%

molybdenum-saturated hSO Surprisingly, with the

exception of vanadate, all other metal ions were also

readily detectable in hSO Although the saturation

lev-els of hSO with copper and arsenite were only 27%

and 23%, respectively, the saturation level for

tung-state was 44% and, for cadmium, 36% (Fig 1A) In

contrast, the levels of MPT saturation in hSO were in

A

B

C

Fig 1 Metal and MPT saturation of purified hSO Purified hSO was analysed after expression from plasmid pTG818 in E coli strain RK5202 (modC)) (A), E coli strain AH69 (moeA)) (B) and

E coli strain RK5206 (mogA)) (C) The following metals were added at a concentration of 100 l M to the growth medium: I,

Na 2 MoO 4 ; II, Cu(NO 3 ) 2 ; III, Na 2 WO 4 ; IV, NaAsO 2 ; V, CdSO 4 ; VI, NaVO3 Dark grey bars, metal contents (l M metalÆl M)1hSO) were determined by ICP-OES (see Experimental procedures) using multi-element standards Light grey bars, the MPT content of hSO was quantified after its conversion to Form A 100% metal or MPT satu-ration is related to a fully active hSO in a 1 : 1 ratio White bars, hSO activity (unitsÆmg)1) defined as an absorbance change of 1.0 AUÆmin)1Æmg)1 protein monitoring the reduction of cyto-chrome c at 550 nm ND, none detectable.

the range 55–74% when cells were grown in the

pres-ence of Na2MoO4, Cu(NO3)2, Na2WO4 or NaAsO2

(Fig 1A) Cells grown in the presence of CdSO4 or

NaVO3 showed lower levels of MPT saturation of

37% and 39%, respectively However, this underlines

the fact that metal-free MPT is inserted into hSO

inde-pendent of the availability of metals

In comparison, the metal insertion pattern was

different in the E coli moeA) strain (Fig 1B) When

using the moeA) strain, neither molybdate nor

tung-state was detected in hSO, showing that both metals

are specifically inserted into MPT by the MoeA

pro-tein In contrast, saturation levels of copper, arsenite

and cadmium were found to be in the region of 21%,

39% and 60%, respectively Again, no vanadate was

detected in hSO Analysis of the uptake of the metal

ions showed that all metals were present in the E coli

extract (data not shown) Thus, E coli is able to take

up vanadate, but vanadate is not inserted into MPT;

this was a rather surprising observation, as vanadium

is known to functionally replace the molybdenum

atom in nitrogenase The MPT saturation of hSO after

expression in the moeA) strain in the presence of

dif-ferent metal ions was comparable, and varied between

71% and 88%

Furthermore, we also examined metal insertion

dur-ing production in the E coli mogA)strain (Fig 1C) It

has been described previously by Miller and Amy [39]

that a molybdate concentration of 1 mm is sufficient to

reverse the mogA) phenotype, restoring nitrate

reduc-tase activity Thus, at a concentration of 100 lm,

neither molybdate nor tungstate reconstituted the

cofactor of hSO In contrast, saturation levels of

copper, arsenite and cadmium were found at 41%,

40% and 65%, respectively As observed after

expres-sion in the modC) and moeA) strains, no vanadate

was detected in hSO The MPT saturation of hSO after production in the mogA)strain in the presence of different metal ions was comparable, and varied between 62% and 73%

Competitive insertion of metal ions into hSO during expression in E coli modC)cells

To analyse the specificity of the insertion of different metal ions into MPT, competition experiments were performed E coli modC)cells were grown in the pres-ence of 100 lm Na2MoO4 and 100 lm Na2WO4, NaAsO2, Cu(NO3)2 or CdSO4 The same competition experiment was performed with Cu(NO3)2 and all other metal ions As shown in Table 1, the MPT satu-ration of hSO was comparable, and varied between 69% and 92% The results in Table 1 clearly show that the presence of equal amounts of molybdate dur-ing growth was sufficient for the specific insertion

of molybdenum into MPT When Na2MoO4 and

Na2WO4, Cu(NO3)2, CdSO4 or NaAsO2 were present during growth, the molybdate saturation in hSO was comparable, and varied between 84% and 70%, whereas only about 5% of the competing metal was inserted (Table 1) This result clearly shows the high specificity of molybdate for insertion into MPT A dif-ferent pattern was obtained for the insertion of tung-state, arsenite or cadmium in the presence of equal amounts of copper during growth When Na2WO4and Cu(NO3)2 were added to the medium, hSO was satu-rated with 48% tungstate and 11% copper; equal amounts of about 30% copper and arsenite were inserted when NaAsO2and Cu(NO3)2 were added, and more cadmium (67.8%) than copper (22.2%) was inserted in the presence of CdSO4 and Cu(NO3)2 in the cell (Table 1) Thus, the affinity of MPT for

Table 1 Activity, metal content and MPT saturation of purified hSO heterologously produced in E coli modC)cells Metals were added dur-ing growth at a concentration of 100 l M each hSO activity (unitsÆmg)1) is defined as an absorbance change of 1.0 AUÆmin)1Æmg)1protein monitoring the reduction of cytochrome c at 550 nm The MPT content of hSO was quantified after its conversion to Form A in comparison with a fully active hSO 100% MPT saturation is related to a fully active hSO in a 1 : 1 ratio Metal contents (l M metalÆl M)1hSO) were deter-mined by ICP-OES (see Experimental procedures) using multielement standards 100% metal saturation is related to a fully MPT-saturated enzyme in a 1 : 1 ratio ND, none detectable.

hSO activity and

metal ⁄ MPT saturation

Metals added to the growth medium

cadmium is higher than that for copper, but, in

gen-eral, there is no preference for the nonspecific insertion

of any other metal ion than molybdate

Analysis of metal insertion into R sphaeroides

dimethylsulfoxide reductase

Most molybdoenzymes in E coli contain the

bis-MGD cofactor For the biosynthesis of bis-MGD, GMP

is attached to the phosphate group of MPT by the

MobA protein [42] It has been shown previously that

MGD formation is catalysed by MobA only after the

coordination of a molybdenum atom to the dithiolene

moiety of MPT [19] To determine whether molybdate

can be replaced by other metals for the biosynthesis

of MGD, R sphaeroides dimethylsulfoxide reductase

was heterologously produced in the presence of

differ-ent metal ions at a concdiffer-entration of 100 lm in the

E coli modC) strain, and analysed for its metal

con-tent Metal analysis of purified dimethylsulfoxide

reductase revealed that only tungstate and molybdate

were inserted into the enzyme (Fig 2) Consistent with

a previous report [43], dimethylsulfoxide reductase

was active in the tungsten-bound form Copper,

cad-mium, arsenite and vanadate were not present in the purified dimethylsulfoxide reductase (Fig 2, data not shown) In addition, the dimethylsulfoxide reductase proteins were devoid of bis-MGD, showing that metal ions such as copper or cadmium cannot replace molybdate in the biosynthesis of MGD, whereas tung-state is able to substitute for this role Thus, GMP is only added to molybdenum- or tungsten-containing MPT

Metal insertion into purified hSO in vitro

It has been shown previously that Moco-free hSO can

be reconstituted with nascent MPT and molybdate

in vitro [44] At the molybdate concentrations used in the assay (> 1 mm), MoeA and MogA were not required for the generation of the active form of Moco, showing that the ATP-dependent activation of MPT and molybdenum is not required for the in vitro ligation of molybdate It was of interest to examine the affinity of free MPT for other metal ions, and

to determine whether MPT chelated with other non-specific metals can also be inserted into hSO in vitro For these experiments, the molybdenum-free human sulfite oxidase Moco domain (hSO-MD) was used as MPT source [45], and extracted MPT was incubated with 100 lm of Na2MoO4, Na2WO4, NaVO3, Cu(NO3)2, CdSO4 or NaAsO2, before purified apo-hSO was added to the mixture As shown in Fig 3, the amount of MPT inserted into hSO was about the same in all incubation mixtures, independent of the added metal, with an MPT saturation in the range 43–47% Analysis of the metal content of hSO revealed that, at a metal concentration of 100 lm, only the bivalent copper and cadmium ions were inserted into hSO (Fig 3A) As shown previously, molybdate is not inserted into MPT at a concentration of 100 lm [44]

As tungstate, arsenite and vanadate were not inserted into MPT at a concentration of 100 lm, higher con-centrations of added metals were analysed However, the addition of these metals at concentrations of up to

1 mm to the reaction mixture did not result in their insertion into MPT (data not shown) In addition, we tested the insertion of metal ions into MPT-containing hSO None of the metals was inserted into hSO (Fig 3B), which is consistent with the previous data for the insertion of molybdate into molybdenum-free MPT-hSO [44], and makes nonspecific binding of the metals to the protein surface unlikely This shows that, after MPT insertion, hSO adopts a conformation that

is not competent for the insertion of metals Thus, any nonspecific metal insertion into MPT has to occur before the insertion of MPT into hSO

Fig 2 Activity, metal and MPT saturation of R sphaeroides

dimethylsulfoxide reductase (DMSOR) Dimethylsulfoxide reductase

was purified after expression from plasmid pJH820 in E coli

RK5202 (modC)) cells The following metals were added during

growth at a concentration of 100 l M : I, Na 2 MoO 4 ; II, Cu(NO 3 ) 2 ; III,

Na2WO4; IV, CdSO4 Dark grey bars, metal contents (l M metalÆl M)1

hSO) were determined by ICP-OES (see Experimental procedures)

using multielement standards Light grey bars, the MPT content of

dimethylsulfoxide reductase was quantified after the conversion

of bis-MGD to Form A 100% metal or MPT saturation was related

to a fully MPT-saturated dimethylsulfoxide reductase in a 1 : 1 or

1 : 2 ratio, respectively White bars, dimethylsulfoxide reductase

activity (unitsÆmg)1) defined as the reduction of 1 lmol of

dimethyl-sulfoxideÆmin)1Æmg)1protein ND, none detectable.

Release of MPT bound to MPT synthase by

metal ions

It has been suggested that, during the biosynthesis of

Moco, the cofactor and its intermediates remain

protein bound until insertion into the specific target

protein, as oxygen seems to be a major factor of free

Moco inactivation [46] Our results showed that metal

ions, such as copper, cadmium or arsenite, might be

inserted nonspecifically into MPT, without the involve-ment of the MogA or MoeA proteins As MPT is pro-duced by MPT synthase, it was of interest to analyse the effect of metal ions on the release of MPT bound

to MPT synthase For our studies, we chose to com-pare the MPT release after metal addition to MPT-containing R capsulatus MPT synthase in comparison with E coli MPT synthase The comparison of the two MPT synthases from different sources was of particu-lar interest, as the phenotype of the R capsulatus moeA) strain has been shown to be repairable with

1 mm molybdate [47], which is not the case for the

E coli moeA) strain [39] Thus, we first analysed the effect of high molybdate concentrations on MPT-satu-rated MPT synthase from the two different sources The results in Fig 4 show that, under the same assay conditions, 85.5% of MPT remained bound to E coli MPT synthase when 1 mm molybdate was added, whereas only 12.3% of MPT remained bound to

R capsulatus MPT synthase This result shows that

E coli MPT synthase binds MPT more tightly; how-ever, the rate of conversion of precursor Z to MPT was the same in both MPT synthases (data not shown)

To analyse the effect of other metal ions on MPT release by MPT synthase, we chose the R capsulatus MPT synthase, which binds MPT less tightly; thus, an effect of metal ions on MPT release should be detected easily R capsulatus MPT synthase was saturated with MPT before the addition of Na2WO4, NaVO3, Cu(NO3)2, CdSO4 or NaAsO2 The incubation

B

A

Fig 3 In vitro reconstitution of hSO with MPT and metal ions (A)

20 l M MPT extracted from hSO-MD was incubated with 100 l M of

Na2MoO4(I), Cu(NO3)2(II), Na2WO4(III), NaAsO2(IV), CdSO4(V) or

NaVO3(VI) for 10 min at 4 C Subsequently, 10 l M of purified

apo-hSO was added to the mixture and incubated for 20 min at 4 C,

before unbound MPT or metal ions were removed by gel filtration.

Dark grey bars, metal contents (l M metalÆl M)1hSO) were

deter-mined by ICP-OES (see Experimental procedures) using

multi-element standards Light grey bars, the MPT content of hSO was

quantified after its conversion to Form A 100% metal or MPT

satu-ration is related to a fully active hSO in a 1 : 1 ratio (B) 40 l M

MPT-containing hSO was incubated with 100 l M of Na 2 MoO 4 (I),

Cu(NO3)2(II), Na2WO4(III), NaAsO2(IV), CdSO4(V) or NaVO3(VI)

for 30 min at room temperature, before unbound MPT or metal

ions were removed by gel filtration Light grey bar, MPT content of

hSO Metal content (l M metalÆl M)1 hSO) of MPT-hSO was below

detection limit ND, none detectable.

Fig 4 Comparison of the effect of 1 m M molybdate on MPT-satu-rated MPT synthase from E coli and R capsulatus 33.2 l M of MPT-saturated MPT synthase from either E coli (I, II) or R capsula-tus (III, IV) was incubated in the presence (II, IV) or absence (I, III)

of 1 m M molybdate The MPT content of MPT synthase was quan-tified after its conversion to Form A.

mixtures were subjected to gel filtration and the MPT

synthase fraction was analysed for its MPT content

The results in Fig 5A show that, under the assay

con-ditions, MPT remained bound to MPT synthase when

metals other than molybdate were added As metal

analysis of MPT synthase revealed that none of the

metals was inserted into MPT (Fig 5A), the dithiolene

group of MPT must be protected in MPT synthase,

and, apparently, only molybdate is able to trigger the

release of MPT This result clearly shows that the non-specific insertion of metal ions into MPT occurs in a step after the release of MPT from MPT synthase The reverse experiment showed that MPT was only bound

to MPT synthase in its metal-free form, and not in the presence of metal ions (Fig 5B)

Discussion

Homeostasis of metal ions is a highly regulated com-plex process in the cell [48] As a defence against metal toxicity, organisms have developed systems for metal detoxification, including specific export systems, as found in E coli [49–51] Our results show that, at high concentrations of metal ions and in the absence of molybdate ions, copper, cadmium and arsenite are inserted into Moco found in hSO In addition to the metals investigated in this report, other metals ions known to bind tightly to sulfur-rich sites, such as zinc and cobalt, are inserted into MPT (data not shown) However, only molybdate and tungstate are specifically inserted into hSO, requiring the catalytic activity of the MoeA protein Although the synthesis of the dioxo Moco found in hSO seems to be more susceptible to nonspecific metal insertion, the biosynthesis of the bis-MGD cofactor present in dimethylsulfoxide reductase provides an additional control step In dimethylsulf-oxide reductase, only molybdate and tungstate are found to be coordinated to bis-MGD, and the inser-tion of either metal results in an active form of dimeth-ylsulfoxide reductase This result shows that MobA is only able to add GMP to the molybdate- or tungstate-substituted form of MPT, and not to other metal-substituted forms of MPT The chemical and physical similarities of tungstate and molybdate are a result of their equal atomic and ionic radii and similar electro-negativity and coordination characteristics, contribut-ing to the discrimination of these metals in biological systems [2,52] In general, tungstate can replace molyb-denum in molybdoenzymes in selected organisms, forming the tungsten cofactor (Wco) [3] Although molybdenum-containing enzymes are found in all aero-bic organisms, tungsten-containing enzymes are gener-ally found only in obligate, typicgener-ally thermophilic, anaerobes Tungsten may have been the first of these elements to be acquired by living organisms However, when the atmosphere became more aerobic, the oxygen sensitivity of tungsten compounds made them less available, and the water solubility of high-valent molybdenum oxides may have become more advanta-geous [3] Because tungsten and molybdenum have similar chemistry, it is possible that, initially, as the transition to an oxygen-rich environment occurred, the

A

B

Fig 5 Analysis of the effect of added metals on MPT binding or

MPT release in purified R capsulatus MPT synthase (A) 33.2 l M

MPT-saturated MPT synthase was incubated with 1 m M of the

indi-cated metal ions, and unbound material was removed by gel

filtra-tion The MPT content of MPT synthase was quantified after its

conversion to Form A (B) 1 m M of metal ions was incubated with

1 m M of MPT prior to the addition of 21.3 l M MPT synthase.

Unbound MPT and metal ions were removed from MPT synthase

by gel filtration The MPT content of MPT synthase was quantified

after its conversion to Form A (A, B) Metal contents were

deter-mined by ICP-OES (see Experimental procedures) using

multi-element standards All metal contents were below the detection

limit 1 m M of Cu(NO3)2, Na2WO4, NaAsO2or CdSO4was added.

ND, none detectable.

latter substituted for the former in enzyme active sites.

The results on dimethylsulfoxide reductase show that

tungsten is still able to replace molybdenum at the

bis-MGD cofactor, resulting in an active enzyme

However, in R capsulatus, the expression of

dimethyl-sulfoxide reductase has been shown to be regulated by

the availability of molybdenum [53]; therefore, it is

unlikely that, during normal growth conditions,

tung-sten replaces molybdenum to produce an active

dimethylsulfoxide reductase in this organism

Our studies also show that bivalent copper and

cad-mium ions and trivalent arsenite ions can be inserted

nonspecifically into MPT without the catalytic activity

of the MoeA or MogA protein Copper and cadmium

also show a higher affinity than molybdate for the

dithiolene group of MPT, as revealed by the in vitro

insertion of metal-substituted MPT into hSO, as these

metals were already inserted into MPT at

concentra-tions of 100 lm Our results show that the nonspecific

insertion of metal ions into MPT occurs at a step

beyond the MPT synthase reaction In MPT synthase,

the dithiolene group seems to be protected in a manner

that makes it inaccessible for the insertion of

nonspe-cific metal ions Only in the presence of high

molyb-date concentration is MPT released and molybmolyb-date

inserted This result may imply that, under conditions

of high molybdate availability in the cell, the activa-tion of MPT by adenylaactiva-tion may not be required, as molybdate is directly inserted into released MPT after its completion by MPT synthase, as shown by the molybdate repairable phenotype of the E coli mogA) strain [39] The comparison of MPT synthase from

E coli and R capsulatus shows that MPT is more eas-ily released from R capsulatus MPT synthase when

1 mm molybdate is added, implying that MPT is less tightly bound in R capsulatus MPT synthase in com-parison with the protein from E coli This result also explains the phenotype of the R capsulatus moeA) strain, which can be complemented by the addition of

1 mm molybdate [47], which is not the case for the

E coli moeA) strain [39] High molybdate concentra-tions result in the release of MPT from MPT synthase, and thus MogA or MoeA are not required under these conditions

Our investigations analysed the insertion of metal ions into MPT beyond the reaction by the MogA and MoeA proteins The insertion of copper, cadmium and arsenite is independent of the MogA and MoeA pro-teins Even if the copper-containing MPT–AMP inter-mediate is formed in E coli, copper can be replaced in this intermediate by other metal ions without the cata-lytic activity of the MoeA protein Our results suggest

Fig 6 Model for the specific and

nonspecific insertion of metal ions

during Moco biosynthesis in E coli.

Specific reactions are marked by full

lines, nonspecific reactions by broken

lines Details of the reactions are given

in the text.

that copper is not required as an intermediate in Moco

biosynthesis in E coli In addition to the known toxic

effects, the toxicity of copper, cadmium and arsenic in

the environment may also be caused by an inhibition

of molybdoenzyme activity in E coli We present a

model for nonspecific metal insertion during Moco

biosynthesis in E coli (Fig 6) Under physiological

molybdate concentrations (1–10 lm), the MogA and

MoeA proteins are required in E coli to form an

MPT–AMP intermediate, facilitating molybdate

inser-tion and Moco formainser-tion in the cell (full lines) Using

the same pathway, tungstate can be specifically

inserted into MPT to form Wco Under high

molyb-date concentrations (> 1 mm), MPT–AMP formed by

MogA is not required and molybdate can be directly

inserted into MPT by the aid of the MoeA protein All

other metal ions, when present at high concentrations,

are inserted nonspecifically into MPT, not requiring

the MogA and MoeA proteins (broken lines)

How-ever, this nonspecific reaction can be outcompeted by

the presence of molybdate, showing that this is the

specific pathway in the cell Nonspecifically formed

Cu–MPT, Cd–MPT or As–MPT can be inserted into

MPT-binding molybdoenzymes, such as sulfite oxidase,

but not into bis-MGD-containing enzymes, such as

dimethylsulfoxide reductase Here, the E coli provides

an additional ‘quality control step’ by the MobA

pro-tein, which only forms the bis-MGD cofactor when

molybdenum or tungsten is inserted into MPT

How-ever, the nature of this quality control step and the

details of bis-MGD formation are not yet known In

our experiments, we were unable to show a

copper-containing MPT–AMP intermediate Thus, for Moco

biosynthesis in E coli, copper is not required and is

rather an inhibitor of molybdoenzymes, inhibiting

enzyme activity when inserted nonspecifically

Experimental procedures

Bacterial strains, plasmids, media and growth

conditions

expression of the R capsulatus moaE and moaD1 genes For

[41] were used for the production of hSO from plasmid

pTG718 [54] R sphaeroides dimethylsulfoxide reductase was

expressed in E coli RK5202 cells from plasmid pJH820 [42]

under the same conditions as hSO Moco-free apo-hSO was

obtained after expression in E coli RK5200 (chlA200::Mu

expressed from pTG818 [54] or pTG718 in E coli RK5202 cells Dimethylsulfoxide reductase, hSO and hSO-MD were purified as described by Temple et al [54] E coli MoaE was expressed from plasmid pGG110 in E coli BL21(DE3) cells (Novagen, La Jolla, CA, USA), cotransformed with plasmid pREP4 (Qiagen, Hilden, Germany) and purified as described previously [55] E coli MoaD was expressed from plasmid pMW15aD in E coli BL21(DE3) cells and purified as described previously [56] Cell strains containing expression vectors were grown aerobically in Luria–Bertani medium at

indi-cated at a concentration of 100 lm

Cloning, expression and purification of

R capsulatus MoaE and MoaD1

DNA fragments containing the coding regions for R

flanking restriction sites were introduced The moaE gene was cloned into the NdeI-BamHI sites of expression vector pET16b (Novagen) and moaD1 into the NdeI-XhoI sites of pET28a (Novagen), resulting in plasmids pSL241 and

transformed with plasmid pSL241 or pMN67 One litre of Luria–Bertani medium was inoculated with 10 mL of an

absor-bance (A) at 600 nm of 0.3–0.5 The expression was induced with 100 lm isopropyl thio-b-d-galactoside, and cells were harvested after an additional growth of 5 h The cell pellet was resuspended in phosphate buffer (50 mm

sev-eral passages through a French pressure cell, and the cleared lysate was applied to 0.5 mL nickel-nitrilotriacetate resin (Qiagen) per litre of culture The column was washed with 20 column volumes of phosphate buffers, one contain-ing 10 mm and the other 20 mm of imidazole Proteins were eluted with buffer containing 250 mm imidazole and, after concentration, the proteins were applied to a PD10 column (GE Healthcare, Munich, Germany) exchanging the buffer for 100 mm Tris, pH 7.2

Metal analysis by inductively coupled plasma-optical emission spectrometry (ICP-OES)

Metal analysis was performed using a Perkin-Elmer Optima 2100DV inductively coupled plasma-optical emission spec-trometer (Perkin-Elmer, Fremont, CA, USA) Protein sam-ples were incubated overnight in a 1 : 1 mixture with 65% nitric acid (Suprapur, Merck, Darmstadt, Germany) at

prior to ICP-OES analysis As reference, the multielement standard solutions XII and XVI (Merck) were used

Moco/MPT analysis

The Moco and MPT contents of purified hSO were

quanti-fied after conversion to Form A-dephospho, as described

previously [57] To determine the MGD content of

for 30 min in the presence of acidic iodine to convert

MGD to Form A Released Form A was detected as

described previously [57]

Enzyme assays

described previously [58] by monitoring the reduction of

cytochrome c at 550 nm, and is defined as an absorbance

change of 1.0 AUÆmin)1Æmg)1protein

assayed as described by McEwan et al [59] with

dithionite-reduced benzyl viologen as the electron donor, and is

defined as the reduction of 1 lmol of dimethylsulfoxideÆ

min)1Æmg)1protein

In vitro incorporation assays

Free MPT was obtained from MPT-containing hSO-MD as

described previously [57] For in vitro metal incorporation

into apo-hSO, 20 lm of extracted MPT was incubated with

20 min, and free MPT and metal ions were subsequently

removed by gel filtration For the determination of metal

incorporation into MPT after insertion into hSO, 40 lm of

with 100 lm of Na2MoO4, Na2WO4, NaAsO2, Cu(NO3)2,

filtration using a Nick column (GE Healthcare), and metal

incorporation into hSO was determined by ICP-OES

analysis

MPT synthase in vitro assays

To determine the release of MPT from MPT synthase,

sepa-rately purified R capsulatus or E coli MoaE and MoaD1

were assembled and incubated with excess MPT prior to the

removal of unbound MPT by gel filtration Subsequently,

After a further incubation step of 10 min, released MPT

was removed by an additional gel filtration step and the

protein fraction was analysed for MPT and metal content

To determine the binding of metal-containing MPT to

addition of 21.3 lm MPT synthase After a further incuba-tion step of 10 min, free MPT and metal ions were removed

by gel filtration and the protein fraction was analysed for MPT and metal content

Acknowledgements

We thank K V Rajagopalan (Duke University, Durham, NC, USA) for helpful discussions, and for providing pTG718, pTG818, pMW15aD and pJH820 This work was supported by Deutsche Forschungs-gemeinschaft Grant LE1171⁄ 3-3 and the Fonds der Chemischen Industrie (FCI)

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