Eur. J. Biochem. 222, 687-692 (1994) 0 FEBS 1994

Isolation of protein FA, a product of the mob locus required for molybdenum cofactor biosynthesis in Escherichia coli Tracy PALMER, Anil VASISHTA, Patrick W. WHITTY and David H. BOXER Department of Biochemistry, Medical Sciences Institute, Dundee University, Scotland

(Received January 24, 1994) - EJB 94 0082/4

The mob mutants in Escherichia coli are pleiotropically defective in all molybdoenzyme activi- ties. They synthesise molybdopterin, the unique core of the molybdenum cofactor, but are unable to attach the GMP moiety to molybdopterin to form molybdopterin guanine dinucleotide, the functional molybdenum cofactor in Escherichia coli. A partially purified preparation termed protein FA (pro- tein factor d’association), is able to restore molybdoenzyme activities to broken cell preparations of mob mutants. A fragment of DNA capable of complementing mob mutants has been isolated from an E. coli genomic library. Strains carrying this DNA in a multicopy plasmid, express 30-fold more protein FA activity than the wild-type bacterium. Protein FA has been purified to homogeneity by a combination of ion-exchange, affinity and gel-filtration chromatography. Protein FA consists of a single polypeptide of molecular mass 22 kDa and is monomeric in solution. N-terminal amino acid sequencing confirmed that protein FA is a product of the first gene at the mob locus. The purified protein FA was required in stoichiometric rather than catalytic amounts in the process that leads to the activation of the precursor of the molybdoenzyme nitrate reductase, which is consistent with the requirement of a further component in the activation.

Molybdoenzymes, with the exception of molybdodinitro- genase, contain a molybdenum cofactor which is composed in its simplest form of a unique pterin derivative, molybdop- terin, to which the molybdenum is bound [l]. The chlorate- resistant mutants of Escherichia coli (recently renamed mo [2]) are pleiotropically defective in all molybdoenzyme activ- ities and are defective in the biosynthesis of active molybde- num cofactor. These mutants carry mutations which map to five distinct regions of the E. coli chromosome: moa, mob, mod, moe and mog. Each of the loci has been cloned and almost all have been sequenced [4-81. The overall functions encoded by most of the loci have been deduced. The moa and moe loci encode proteins which assemble the molybdopterin portion of the cofactor [9, 101. The mod locus encodes the molybdate uptake system of the cell [9, 111. The function of mog is not known [12]. For several years the role of the mob locus in cofactor biosynthesis was unclear since these mu- tants possessed molybdopterin [13, 141. However, recently it has been demonstrated that in E. coli the active form of the cofactor is molybdopterin guanine dinucleotide (MGD), a covalent complex of molybdopterin with guanosine mono- phosphate. Mutants defective at mob are unable to attach GMP to molybdopterin, suggesting that the products of the mob locus are responsible for this late step in molybdenum cofactor biosynthesis [15].

Correspondence to D. H. Boxer, Department of Biochemistry, Medical Sciences Institute, Dundee University, Dundee, Tayside, Scotland, DD1 4HN

Fax: +44 382 322558. Abbreviations. MGD, molybdopterin guanine dinucleotide ; pro-

Enzyme. Nitrate reductase (EC 1.7.99.4). tein FA, protein factor d’association.

The biochemical analysis of molybdenum cofactor bio- synthesis has exploited the observation that the incubation of crude soluble extracts of moa and mob mutants leads to the formation of active molybdoenzymes [14]. The activation of molybdoenzymes such as nitrate reductase, is a convenient way to follow the synthesis of molybdenum cofactor in vitro since such enzymes are inactive unless the appropriate form of the cofactor is present. In previous work [17] the soluble cell extract of a moa mutant was fractionated by ion-ex- change and gel-filtration chromatography to obtain a 16fold purified preparation, termed protein FA, which when added to the soluble fraction of a mob mutant brought about the activation of the soluble precursor of the molybdoenzyme, nitrate reductase. Protein FA activity is absent from mob mu- tants [18].

In this paper we report the cloning of a mob-complement- ing E. coli DNA fragment, which when present on a multicopy plasmid results in a 30-fold overexpression of pro- tein FA activity. Protein FA has been purified to homo- geneity.

MATERIALS AND METHODS Bacterial strains

Table 1. The E. coli strains employed in this study are listed in

Isolation of DNA able to complement mob mutants An E. coli genomic DNA library in AD69 containing

DNA fragments prepared by Sau3A partial digestion, was a gift from Professor N. Murray. From this library, the clone,

688

Table 1. Strains, phages and plasmids used in this study.

Name Genotype Origin

AP24 RK5208 SE.5000 iPW69 pPW696

F- thr leu his pro arg thi ade gal lacy malE xyl ara mtl str Tlr2 mob24 AlacU169 araD139 rpsL gyrA non mob207: : mucts

[I61 [I21

F- AlacU169 araD139 rpsL1SO relAI deoCl ptsF rbsRJzbB recAS6 M. Berman iBam il" A(sr1 I-srlA2) imm21 nin5 shn6" mob' this study pBR322 mob+ (AmpTeP) this study

APW69, was isolated which complemented to nitrate reduc- tase sufficiency, the mob Mucts insertion mutant, RK5208. This clone also complemented a further four independent mob Mucts insertion mutants and the point mob mutation present in AP24, the strain that we have used for biochemical analysis of the mob defect [14, 191. The mob-complementing fragment of DNA (approximately 1.5 kb) was released from APW69 and cloned into the BamHI site of plasmid pBR322. The resulting plasmid, pPW696, was transformed into the recA host strain, SE5000. Restriction analysis of the cloned fragment (data not shown) was consistent with its being the same as the BamHI fragment cloned by Reiss et al. [3] which has been partially sequenced [8].

Growth of bacteria and preparation of subcellular fractions

The bacteria were grown in a basal medium [20] with the addition of glucose (2 g/l), yeast extract (Difco) (2 gA), Bactopeptone (Difco) (2 gfl) and 1 pM sodium molybdate. For expression of nitrate reductase, KNO, (1 g/l) was added. Anaerobic growth was accomplished in capped aspirator jars filled almost to the top. Cultures were incubated at 37"C, unless the strain was a bacteriophage Mu (Mucts) lysogen, when the temperature was kept to 30°C. Cells were harvested at late exponential phase and suspended after washing in 50 mM Tris/HCl pH 7.6, 1 mM benzamidine HC1. The cells were either used immediately or stored as pellets at -20°C until required. Bacteria were broken by passage through a French press [21]. The crude cell extract was obtained following the removal of cellular debris (centrifugation at 18000 X g for 20 min). The cell supernatant was prepared by further centrifugation (120000 X g for 90 min). All steps were performed at 4°C.

Liquid culture of bacteria carrying pPW696 was per- formed in the presence of 50 pg/ml ampicillin.

Purification of protein FA

10 g (wet mass) of aerobically grown SE 5000 containing pPW696 was resuspended in 50 mM Hepes/NaOH pH 7.6, 1 mM benzamidine/HCl, disrupted and the soluble fraction prepared as described above. Cells were grown aerobically since we have shown previously that the level of protein FA activity is maintained at the anaerobic level during aerobic growth [18]. In addition, 1OmM sodium tungstate was in- cluded in the growth medium in order to ensure that any expressed nitrate reductase was inactive and would not sub- sequently interfere in the assay of protein FA activity [22]. All dialysis and purification steps were performed in buffer A: 50 mM Hepes/NaOH pH 7.6, 1 mM benzarnidineMC1, 1 mM EDTA, 5 mM 2-mercaptoethanol. For gel filtration steps, buffer A was routinely supplemented with 0.25 M

NaC1. Samples were concentrated for gel filtration by centrif- ugation through an Amicon Centricon 10 microconcentrator.

Protein FA activity assay The assay of protein FA activity was based on that de-

scribed earlier [17, 181. The method monitors the activation of the inactive precursor of nitrate reductase in a crude ex- tract prepared from a mob strain grown anaerobically with nitrate. This activation requires the synthesis of an active form of the molybdenum cofactor. Cells of the mob strain AP24 were grown anaerobically in the presence of nitrate, and the crude supernatant fraction prepared. Aliquots of frac- tions to be assayed were added to 200 pl of the AP24 super- natant fraction and the final volume adjusted to 400 pl with buffer A. The samples were flushed with N,. All operations were performed at 0°C. The assay mixtures were incubated at 37°C for 10 min, after which they were placed on ice which effectively stopped the reaction. Aliquots of the reac- tion mixture were assayed for nitrate reductase activity.

Nitrate reductase activity was measured discontinuously by estimation of the rate of nitrite production from nitrate in the presence of reduced benzylviologen [23]. Activation of nitrate reductase was always confirmed to be incubation- time-dependent and proportional to the amount of protein FA added. Protein concentration was determined by the method of Lowry [24].

Polyacrylamide gel electrophoresis Polyacrylamide gel electrophoresis in the presence of

SDS was carried out using the TrisiTricine buffer system de- scribed by Schagger and von Jagow [25]. The resolving gel was 10% (masdvol.) acrylamide, 0.6% (mass/vol.) bisacrylamide. For N-terminal amino acid sequencing, pro- teins were resolved electrophoretically by the method of Laemmli [26], electroblotted on to a nitrocellulose membrane and analysed using a Beckman 470A gas-phase automatic peptide sequencer as described previously [4].

RESULTS Overexpression of protein FA activity

After transformation of E. coli strain SE5000 with the mob+ plasmid, pPW696, expression of protein FA activity was greatly increased. This is shown in Fig. 1. The protein FA activity from the overexpressing strain eluted on gel filtration in the same position as the protein FA activity from the untransformed wild-type strain. When the strain was transformed with the plasmid vector (pBR322) only normal expression of activity was seen. Comparison of the areas in- cluded by the protein FA activity elution profiles in Fig. 1 indicates that the cells containing the mob+ plasmid express

- - E o ! . / - % - m 1 8 0

6 0 1 0 0 1 4 0

Eluate volume (ml)

Fig. 1. Transformation of E. coli with plasmid pPW696 leads to overexpresslon of protein FA activity. Crude cell extracts of E. coli strain SE5000 (+), SE5000 transformed with the plasmid vector pBR322 (0) and SE5000 transformed with the recombinant mub+ plasmid, PPW696 (El) were chromatographed on a Sephadex G-100 column. This separates protein FA from endogenous nitrate reduc- tase activity which elutes in the void volume and which would in- terfere with the protein FA activity measurements. The column frac- tions were assayed for protein FA activity as described in Materials and Methods.

about 30-fold more protein FA activity. This level of overex- pression presumably reflects the high copy number of the plasmid and strongly indicates that protein FA is a product of the mob locus. The overexpressing strain was used for the subsequent purification of protein FA.

Purification of protein FA The soluble fraction from l o g (wet mass) of SE5000

transformed with pPW696, was dialysed twice for 1 h against a large volume of buffer A. Initial fractionation and concen- tration was carried out on a 15-ml DEAE-cellulose (DE52) ion-exchange column which had been pre-equilibrated with the same buffer. Protein FA activity was eluted in a concen- trated state following the passage of 0.2 M NaCl in buffer A through the column. Fractions with activity were combined and dialysed against buffer A at 4°C for 16 h.

The sample was applied to a 10-ml DEAE-Sepharose CL6B column, again pre-equilibrated with buffer A. Bound protein was eluted with a linear gradient of 0-0.3 M NaCl in buffer A. Protein FA activity eluted at approximately 0.15 M NaCl, broadly as reported by Rivikre et al. [17]. The active fractions were combined and dialysed as before. All of the previous steps were carried out at 4°C. The remaining stages of purification were performed at room temperature.

The sample was chromatographed on Cibacron blue 3GA column, linked to a Pharmacia FPLC system. Although al- most all of the protein passed straight through the column protein FA remained bound to the resin. A 0-0.5 M gradient of NaCl eluted further protein from the column and a further step of 2 M NaCl displaced protein FA.

The final purification step was achieved by FPLC Su- perose 12 gel filtration in buffer A supplemented with 0.25 M NaC1. Protein FA eluted as a single coincident peak of protein and activity corresponding to an apparent molecu- lar mass of 22 kDa, as shown in Fig. 2.

Fig. 3 shows the polypeptide composition of active frac- tions at each stage of the purification as analysed by SDSI

0.50

E, 0 OD cv - 8 0.25 m e B n U

0 I I

1 0 1 5

Eluate volume (ml)

Fig. 2. FPLC gel filtration of protein FA. The combined fractions with protein FA activity following the Cibacron blue chromatogra- phy were concentrated to 200 p1 and applied to a Superose 12 col- umn pre-equilibrated with buffer A containing 0.25 M NaCl. Frac- tions of 250 p1 were collected and assayed for protein FA activity.

t 2 3 4 5

-46 kDa - 3 0

-21.5

- 14.3

‘6.5 Fig.3. Preparation of protein FA: SDSPAGE analysis of the protein FA active fractions. Electrophoresis was performed using the system of Schagger and von Jagow [25] as described in Materials and Methods. Lane 1, soluble fraction, (3 mg protein); lane 2, com- bined protein FA active fractions after DEAE-cellulose concentra- tion (500 pg protein); lane 3, combined protein FA active fractions after DEAE-Sepharose chromatography (200 pg protein) ; lane 4, combined protein FA active fractions after Cibacron blue chroma- tography (50 pg protein); lane 5, peak protein FA active fraction after Superose 12 gel filtration (4 pg protein).

PAGE. After the final chromatographic step, the protein FA preparation revealed a single polypeptide of apparent molec- ular mass 21.5 kDa. Table 2 outlines the yield of protein FA and the level of purification observed after each step. The overall purification approached 2000-fold. The best single purification step was the Cibacron blue column which gave a 30-fold purification. The assay procedure involves the use of a mob crude extract and the absolute values for the protein FA activity depend on the state of the extract. Accordingly, the activities of the combined fractions at each stage were reassayed simultaneously with the same mob crude extract at the completion of the preparation in order to make the best comparison between fractions. However, comparison of the SDSPAGE analysis of the preparation before and after the Superose 12 gel filtration suggests that this step achieved more than the five-fold purification indicated by the activity measurements. This may reflect the uncertainty of the activ- ity assay or that protein FA stains rather less well with Coo- massie blue than other proteins. Laser desorption mass spec- trometry of the peak active fraction after Superose 12 gel

690

Table 2. Purification of protein FA. Specific activity is in arbitrary units.

Total protein Specific activity Activity yield Purification Stage

mg Cell extract 2241 DEAE-Sepharose 168 Cibacron blue 2.0 Superose 12 0.5

0.88 10.6

306 1596

%

100 80 50 40

-fold 1

12 350

1816

filtration gave a single species with molecular mass of 21.8 kDa (not shown). The margin of error on this measure- ment is ? 1 %.

N-terminal amino acid sequence Analysis of the published partial DNA sequence for mob

[4] suggests a single polypeptide translated from an ATG start codon of N-terminal sequence MTTI. However, the N- terminal five amino acids of the purified protein FA after the Superose 12 gel filtration were sequenced and found to be MNLMT. This indicates that the alternative GUG start, three codons upstream, is used. This confirms directly that protein FA is a product of the mob locus. This is entirely consistent with its ability to restore nitrate reductase and other molyb- doenzyme activities when incubated with crude extracts of mob mutants. In E. coli GUG can encode formylmethionine and functions as the start codon in approximately 10% of E. coli proteins [27]. GUG when present internally codes for valine.

Properties of purified protein FA The purified protein FA was stable following aerobic

storage in solution at 4°C for several weeks. Furthermore, it could be stored frozen at -20°C without loss of activity for 14 days. The activity was unaffected after prolonged dialysis.

The time-dependent activation of nitrate reductase in the crude extract of a mob mutant, brought about by purified protein FA, is shown in Fig. 4A. Also shown in the same figure is the activation curve obtained when purified protein FA was replaced by an aliquot of the cell supernatant of strain SE5000 (containing pPW696). It is clear that the acti- vation of nitrate reductase by pure protein FA and by the cell supernatant followed similar kinetics. Furthermore, approxi- mately the same overall level of activation was achieved. In Fig. 4B, cell extracts of the mob mutant were incubated with a range of concentrations of purified protein FA. In agreement with the observations of Low et al. [18], increas- ing the amount of protein FA in the activation mixture led to an increase in the rate of activation of nitrate reductase but did not affect the extent of the activation.

DISCUSSION This report describes the purification of E. coli protein

FA to homogeneity and confirms it unequivocally to be a product of the mob locus. The very low amount of this pro- tein present in wild-type cells has previously hindered attempts at purification [17]. This problem has been over- come by the use of a strain which carries the mob locus on a multicopy plasmid and which expresses a 30-fold increased level of protein FA activity over that of wild-type cells. It

A

2oo I

0 2 0 40 6 0 80 100

Reconstltution t h e (min) B 200 I

100

0 0 2 0 4 0 6 0 8 0

Reconstitution time (min)

Fig. 4. Activation of mob nitrate reductase in crude cell extracts by purified protein FA. Conditions for the activation procedure were as described in Materials and Methods. The mob crude cell extract was prepared from strain AP24. (A) (El) Crude cell extract of SE5000 containing pPW696 (40 pg protein) added to 200 pl mob crude cell extract in a final volume of 400 pl; (4) purified protein FA (approximately 1 pg protein) added to 200 p1 mob crude cell extract in a final volume of 400 pl. (B) Activation of mob nitrate reductase by a range of concentrations of purified protein FA. Pro- tein FA was present in the following amounts: (+) 600ng; (El) 300 ng; (0) 60 ng; (0) 30 ng in a final volume of activation mixture of 400 pl.

should be noted that even in overexpressing strains, it is still not possible to distinguish protein FA as a protein-stained band after SDSPAGE of cell extracts. This is consistent with a further 2000-fold purification being required to achieve homogeneity. It is clear that protein FA constitutes a very small proportion (< 0.001 %) of the soluble cellular protein in normal cells. Analysis of gene fusions in which j?-galac-

691

tosidase expression is placed under mob control, has also demonstrated that mob expression is extremely weak [28].

The low cellular content of protein FA suggests that it performs a catalytic role. However, attempts to demonstrate that protein FA brought about the activation of mob nitrate reductase in a catalytic manner were unsuccessful. The best estimates, calculated from experiments such as those de- scribed in Fig. 4B, indicate that about three molecules of nitrate reductase can be activatedmolecule of protein FA. Such calculations necessarily assume that the specific activ- ity of the activated nitrate reductase is the same as that re- ported for the most active purified preparations of the en- zyme, i.e. 75 pmol nitrate reduced X min-' X mg protein-' [29]. Given the assumption of this calculation, this approxi- mates to a stoichiometry of close to 1 : 1. The possibility that the majority of the purified protein was inactive is unlikely given its overall stability and the high yield of activity achieved in the purification. More likely another component required for the nitrate reductase activation assay is present in limiting quantities in the mob crude extract. Protein FA activity is susceptible to exposure to proteinases [30]. In or- der to limit any loss of protein FA activity during the activa- tion by proteases present in the crude extract, a cocktail of protease inhibitors was added to the suspension of mob cells before disruption. This did not significantly affect the result.

Recently as part of the E. coEi genome sequencing pro- ject, the DNA sequence of the region covering the mob locus has been reported [31]. The sequence completely confirmed the partial mob sequence [8] which we show here encodes protein FA. However, a second open reading frame immedi- ately downstream of the protein FA gene was revealed. It is clear therefore, that protein FA is encoded by the first gene of the mob operon, mobA. The significance of the putative second gene remains to be established. The predicted molec- ular mass of protein FA, calculated from the DNA sequence of mobA,is 21638Da. This is in very close agreement with the experimentally determined values that we report. We show that protein FA purified as described here exists as a monomer in solution. This is in agreement with an estimated molecular mass for active protein FA of 18 kDa, obtained following gel filtration of partially purified extracts of wild- type cells [30].

Protein FA was purified by a combination of anion-ex- change, affinity and gel-filtration column chromatography. The most effective purification step was achieved using a Cibacron blue agarose column. Cibacron blue is one of a series of dye ligands whose active group is structurally simi- lar to that of purine nucleotides [32]. Interestingly, protein FA could be recovered from the affinity column by elution with 10 mM GTP. The significance of these observations however is not clear since there is no indication from inferred protein sequence analysis that protein FA contains a nucleo- tide binding motif [8, 331. We have shown previously that the protein-FA-dependent activation of nitrate reductase from a mob strain requires GTP [19]. Whether protein FA interacts directly with GTP is presently being investigated.

Pure protein FA brings about the activation of nitrate re- ductase in crude cell extracts of mob strains. The kinetics of nitrate reductase activation were comparable to those ob- served with crude preparations of protein FA. The pure pro- tein FA does not, however, activate the nitrate reductase in the assay system in a catalytic manner. The protein-FA-de- pendent activation process must reflect the mechanism by which molybdopterin guanine dinucleotide (MGD) is synthe- sised. The process requires GTP [19] and the inactive nitrate

reductase precursor present in mob strains has bound molyb- dopterin [19]. Covalent attachment of GMP from GTP to enzyme-bound molybdopterin is thought to occur which then brings about the activation of nitrate reductase [15]. Consis- tent with this interpretation, purified nitrate reductase precur- sor from a mutant has been activated in vitro where the only source of molybdopterin that could be detected was present in the inactive nitrate reductase precursor. This activation is dependent upon protein FA but a further protein factor is also required [19]. The failure to observe catalytic activation of mob nitrate reductase by pure protein FA may reflect the requirement for this additional component. The nature of this additional protein and whether it affects the stoichiometry of nitrate reductase activation is currently under investigation.

We wish to thank M. Berman and N. Murray for the gift of strains and the AD69 E. coli genomic library. B. Dunbar is thanked for performing the mass spectrometry of protein FA. This work is supported by the Science and Engineering Research Council (GW H10559). We thank G. Giordano for many helpful discussions.

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