©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biotin Synthase from Escherichiacoli, an Investigation of the Low Molecular Weight and Protein Components Required for Activity inVitro(*)

(Received for publication, January 9, 1995; and in revised form, May 9, 1995)

Olwen M. Birch Martin Fuhrmann Nicholas M. Shaw (§)

From theBiotechnology Department, Lonza A.G., CH-3930 Visp, Switzerland

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have developed a radiochemical method for the measurement of biotin synthase activity in vitro. A cell-free extract from an Escherichia coli strain containing a cloned bioB (biotin synthase) gene was incubated with [^14C]dethiobiotin, which was converted to [^14C]biotin. The assay was used to identify the low molecular weight compounds and two of the proteins that, in addition to the bioB gene product, are required for biotin synthase activity in vitro. The low molecular weight compounds are cysteine; S-adenosylmethionine; thiamine pyrophosphate; Fe; a pyridine nucleotide (the most effective being NADPH); and one of the amino acids asparagine, aspartate, glutamine, or serine. The proteins are flavodoxin and ferredoxin (flavodoxin)-NADP reductase (EC 1.18.1.2). A third thiamine pyrophosphate-dependent protein is also required for activity. When the cell-free extract was incubated with nonlabeled dethiobiotin and either [S]cysteine or [S]cystine, S was incorporated into biotin, and we present further evidence that cysteine, and not S-adenosylmethionine or methionine, is the sulfur donor for the biotin synthase reaction.


INTRODUCTION

The vitamin biotin is synthesized by microorganisms (1) and plants (2) . It is an essential cofactor for carboxylase-catalyzed reactions, where it is bound through its side chain carboxyl group via an amide bond to the -amino group of a specific enzyme lysine residue, and functions by carrying activated carbon dioxide on the N-1` atom(3) . Biotin is both of great commercial and scientific interest. It is sold as a pharmaceutical and as a food, feed, and cosmetic additive. It is synthesized chemically on an industrial scale by multistep processes (4, 5) , but intensive research is also being carried out into developing a microbiological production method(6) . In addition, the pathway in plants is a potential target for herbicide action(2) .

The biotin biosynthetic pathway in bacteria, especially that of Escherichia coli and Bacillus sphaericus, has been widely investigated(7, 8) . The steps leading to the formation of pimeloyl-CoA in E. coli are still unclear(9, 10) , but the pathway from pimeloyl-CoA to biotin is well understood, with the exception of the last step, the conversion of dethiobiotin to biotin. This reaction involves the insertion of a sulfur atom between two unsaturated carbon atoms to form the thiophene ring of biotin and is catalyzed by biotin synthase, the product of the bioB gene(11) . Similar enzyme-catalyzed reactions involving sulfur insertion occur in the synthesis of isopenicillin N(12) , lipoic acid (13) , and thiamine(14) . The mechanism by which unactivated C-H bonds in these compounds are converted to C-S bonds is of considerable interest. It is only in the last 2 or 3 years, with the introduction of assay methods in vitro, that detailed investigation of biotin synthase has been possible(15, 16, 17) . Experiments to try to elucidate the mechanism of the reaction and to identify possible intermediates had until then been carried out with whole cells. The results obtained from such experiments were not always conclusive, because possible precursors or intermediates for the reaction could be extensively metabolized within the cell before incorporation into biotin(18) . Nevertheless it had been established that the biotin synthase reaction may proceed by way of a radical mechanism(19) , and that hydroxylated intermediates or intermediates with carbon-carbon double bonds are probably not involved(20) .

The origin of sulfur in biotin has been investigated in a number of microorganisms, using whole cells, and various sulfur donors have been proposed. It has been established that in E. coli the sulfur atom in biotin is derived from cysteine, but one or more steps may be involved between cysteine and the direct sulfur donor for the reaction(21, 22) . A recent report in which cysteine is shown to act as sulfur donor for the biotin synthase reaction in vitro confirms the involvement of cysteine, but it does not rule out the possibility of further metabolism of the cysteine before incorporation into biotin(23) . The identity of the direct sulfur donor for biotin synthase therefore remains unknown and can only be established using a defined assay system for biotin synthase in vitro.

Our group aims to produce biotin on an industrial scale by fermentation, and to this end we have cloned the bio operon from E. coli and overexpressed the biosynthetic enzymes (17) . We found that the biotin synthase step was limiting for our process, and consequently, we have focussed our attention on this reaction. When we started this work, no assay method was available for biotin synthase in vitro, despite years of work by many research groups. The original aim of our research was to develop an assay for biotin synthase in vitro, to identify the direct sulfur donor for the reaction, and to purify and characterize the enzyme. We modified our third aim when we found that the enzyme requires a number of low molecular weight compounds and proteins for activity in vitro. It then became of interest to us to identify all of the components of the enzyme system in case this knowledge could be used to improve our biotin fermentation process. These experiments were also an important prerequisite for detailed kinetic and mechanistic studies. This paper describes our findings.


MATERIALS AND METHODS

Bacterial Strains and Plasmids

The work described here was carried out using E. coli BM4062(24) , containing the plasmid pBO30A-15/9, which carries the genes bioBFCDAOrf1, or E. coli XL1-Blue (25) containing the plasmid pBO74DeltaB, identical to pBO30A-15/9 but with the bioB gene deleted (17) . The strains and extracts derived from them will be referred to as ``biotin synthase plus'' and ``biotin synthase minus,'' respectively. The host strains both carry the wild-type chromosomal genes for biotin synthase, but these are repressed under the conditions of our experiments.

Chemicals

Dethiobiotin was chemically synthesized by Dr. Laurent Duc, Lonza AG. [^14C]Dethiobiotin (57.2 mCibulletmmol) was custom synthesized by Isotopchem, Ganagobie, France. D-[carbonyl-^14C]Biotin, (50-60 mCibulletmmol), L-[S]cystine (100.3 mCibulletmmol), and L-[S]cysteine (>600 Cibulletmmol) were from Amersham Corp. L-[S]Methionine (>1000 Cibulletmmol) was from DuPont NEN. All other chemicals were either from Sigma or Fluka and were the purest grade available.

Growth of Cells

Cells were grown at 37 °C in nutrient yeast broth (NYB) medium (nutrient broth no. 2, Oxoid, 25 gbulletliter; yeast-extract, Oxoid, 5 gbulletliter) with the addition of chloramphenicol (20 µgbulletml) for plasmid stability. Cells were grown on both small scale in shake flasks, and in fermenters of up to 800 liters.

Preparation of Cell-free Extracts

Cells were harvested in the exponential phase and concentrated by filtration and centrifugation. After washing in 100 mM Hepes buffer, pH 7.5, they were resuspended to a cell density equivalent to an absorbance of 500 (measured spectrophotometrically at 600 nm) and broken open with a continuous cell homogenizer (Stansted Fluid Power Ltd., Stansted, United Kingdom, model LD10-30-P-C). Cell debris was removed by centrifugation at 20,000 g for 30 min, and the resulting cell-free extract was stored at 80 °C.

Preparation of a Cell-free Extract for the Identification of the Low Molecular Weight Compounds Required for Biotin Synthase Activity in Vitro

A cell-free extract from the biotin synthase plus strain, free of low molecular weight components, was prepared by desalting either once or twice on Sephadex G-25 M gel-filtration columns (Pharmacia Biotech Inc., PD-10). When necessary, extracts were concentrated after desalting by ultrafiltration using Centricon 10 microconcentrators (Amicon).

Measurement of Biotin Synthase Activity in Vitro

To assay biotin synthase activity in vitro, the incorporation of radioactive label from either [^14C]dethiobiotin, [S]cystine or [S]cysteine into biotin was measured in the presence of a cell-free extract or protein fraction. A typical standard assay, with a biotin synthase plus cell-free extract, including all of the cofactors that we know to be essential for the reaction, was as follows: cell-free extract (typically 50 µl, 2.75 mg of protein or 100 µl, 5.5 mg of protein) was incubated in a final volume of 250 µl at pH 7.5 with Fe-gluconate (50 nmol), NADPH (25 nmol), thiamine pyrophosphate (25 nmol), S-adenosylmethionine (23 nmol), asparagine (3.75 µmol), dithiothreitol (250 nmol), Hepes buffer (25 µmol), and either [^14C]dethiobiotin (0.1 µCi, 1.95 nmol) plus cysteine (83 nmol), or nonlabeled dethiobiotin (23 nmol) plus [S]cysteine (20 µCi, 1.32 nmol). All compounds except the labeled materials were prepared in Hepes buffer. After incubation at 37 °C for 1 h, the reaction was stopped by the addition of 250 µl of 12.5% (w/v) trichloroacetic acid. Precipitated protein was removed by centrifugation, and the supernatant was loaded on to a disposable C(18) solid phase extraction column (Macherey-Nagel, Chromabond C(18) ec, 100 mg) that had been equilibrated with methanol (1 ml), water (1 ml), and 1% (v/v) acetic acid (1 ml). After washing with 1% (v/v) acetic acid (1 ml) and water (1 ml), [^14C]dethiobiotin and [^14C]biotin or [S]biotin were eluted with 0.5 ml of methanol. The samples were dried under vacuum and resuspended in buffer suitable for TLC (^1)or HPLC analysis.

Samples for semiquantitative analysis by TLC and autoradiography were resuspended in 20 µl of methanol/water/acetic acid (65:25:10), and 2.5 µl was loaded on to a silica gel high performance TLC plate (E. Merck, Darmstadt, Germany). TLC plates were developed with chloroform/methanol/formic acid (17:3:0.2) and then exposed to x-ray film (beta-max, Amersham Corp.) for autoradiography.

Samples for quantitative analysis were resuspended in 50 mM NaH(2)PO(4), pH 3.5, containing 20 mM triethylamine and 203 mgbulletliter each of dethiobiotin and biotin as carrier, and analyzed by reverse-phase HPLC using a Hewlett-Packard ODS Hypersil, 2.1 mm 200 mm, 5-µm particle size column. The eluent was 10% (v/v) acetonitrile in 50 mM NaH(2)PO(4), pH 3.5, containing 20 mM triethylamine, and the column was at 35 °C. Dethiobiotin and biotin were detected at 191 nm, and radioactivity was measured with a Berthold LB 506 C-1 on-line radioactivity detector equipped with a Z-200 (200 µl volume) cell and Winflow radiochromatography software. The flow rate through the column was 0.35 mlbulletmin and the total flow through the cell was 1.75 mlbulletmin, including the scintillator (Quickzint Flow 303, Zinsser Analytic, Maidenhead, UK).

In addition to the TLC and HPLC methods described above, different TLC and HPLC eluents were used to confirm the identity of the radioactive biotin produced in the assays. For TLC, butanol-1/water/acetic acid (65:25:10) was the eluent with the silica-gel high performance TLC plates. For HPLC, a paired-ion method was used. The column was the same as that used for the reverse-phase method, but the eluent was 80 mM KH(2)PO(4), pH 6.5, plus 4.2 mM tetrabutyl ammonium phosphate and 16% (v/v) methanol. The column was at 40 °C.

N-terminal Amino Acid Sequencing

This was by Edman degradation with either a model 473A or a model 477A Microsequencer (Applied Biosystems) equipped with Pro-blott reaction cartridges. Preparation of the sample of flavodoxin for sequencing was as described in (26) , except that for the purification of the tryptic peptides the HPLC method at pH 2 was used before that at pH 7.

SDS-Polyacrylamide Gel Electrophoresis and Western Blotting

Protein samples were analyzed by SDS-PAGE either by the method described in (27) , or with a Pharmacia Phast System. SDS-PAGE and Western blotting for sequencing were carried out using the methods described in (28, 29, 30) . Polyvinylidine difluoride membranes were stained with Amido Black and destained with water. Bands of interest were excised, dried at room temperature, and stored at 4 °C before microsequencing.

Preparation of Enriched Protein Fractions

The enriched protein fractions described below were not concentrated to a specific protein concentration, but they were prepared so that 50 µl of each protein fraction gave a clear signal in the in vitro assay. The volumes of the protein fractions used are specified for each assay.

Preparation of Enriched Biotin Synthase and Enriched Biotin Synthase Plus Thiamine Pyrophosphate

A 100,000 g supernatant, prepared from a biotin synthase plus cell-free extract was diluted to 10 mgbulletml of protein with 20 mM Tris buffer, pH 7.5, containing 1 mM dithiothreitol. The extract (500 ml, 5 g of protein) was loaded on to an anion-exchange column (Pharmacia Q Sepharose Fast Flow, 50-mm diameter 125-mm height, 245-ml bed volume) equilibrated with the same buffer. Biotin synthase was eluted with a linear NaCl gradient (0-600 mM) in the Tris/dithiothreitol buffer in 1500 ml. The flow rate was 5 mlbulletmin. Biotin synthase eluted in the position shown in Fig.1. Fractions were tested for biotin synthase activity using a modified standard assay, in which a biotin synthase minus cell-free extract and the fractions to be assayed replaced the biotin synthase plus extract. Fractions with the highest biotin synthase activity were pooled, concentrated by ultrafiltration, and stored at -80 °C. Biotin synthase plus thiamine pyrophosphate was prepared by the same method, except that thiamine pyrophosphate (1 gbulletliter) was added to the buffers, and all fractions with biotin synthase activity were pooled, including the fractions with weaker activity that eluted after the main peak, as these fractions contained the thiamine pyrophosphate-dependent protein.


Figure 1: Preparation of enriched protein fractions by Q Sepharose Fast Flow anion-exchange chromatography. A 100,000 g supernatant, prepared from a biotin synthase plus cell-free extract, was diluted to 10 mgbulletml of protein with 20 mM Tris buffer, pH 7.5, containing 1 mM dithiothreitol and 1 gbulletliter thiamine pyrophosphate. The extract (500 ml, 5 g of protein) was loaded on to an anion-exchange column (Pharmacia Q Sepharose Fast Flow, 50-mm diameter 125-mm height, 245-ml bed volume) equilibrated with the same buffer. The flow rate was 5 mlbulletmin, and the fraction size was 10 ml. The elution positions of the proteins are indicated by the horizontallines: 1, ferredoxin (flavodoxin) NADP reductase; 2, biotin synthase; 3, thiamine pyrophosphate-dependent protein; 4, flavodoxin. The bullet--bullet line shows the NaCl gradient.



Preparation of Enriched Fractions of Biotin Synthase Accessory Proteins

Our results have shown that at least three additional proteins are required for biotin synthase activity in vitro. We describe these as the biotin synthase accessory proteins. Two methods were used for the preparation of enriched fractions of these accessory proteins. Initially, ammonium sulfate fractionation was used, but while purifying these proteins, it became apparent that they could be separated by anion-exchange chromatography (Q Sepharose Fast Flow, Pharmacia). The accessory proteins and biotin synthase could therefore be purified from the same cell-free extract, as they were separated in the first chromatographic step (Fig.1). In the later stages of this work, enriched flavodoxin and ferredoxin (flavodoxin) NADP reductase preparations were routinely obtained from biotin synthase plus cell-free extracts using the chromatographic method.

Preparation of Enriched Flavodoxin by Ammonium Sulfate Precipitation

Biotin synthase minus cell-free extract was brought to 55% saturation with ammonium sulfate, and the precipitate was removed by centrifugation. The flavodoxin-containing supernatant was desalted into 100 mM Hepes buffer, pH 7.5 (Sephadex G-25M PD-10 gel-filtration column, Pharmacia), concentrated, and stored at -80 °C.

Preparation of the 0-45% Ammonium Sulfate Fraction

A biotin synthase minus cell-free extract was precipitated with 45% saturation of ammonium sulfate. The precipitated protein was resuspended in 100 mM Hepes buffer, pH 7.5, desalted, (Sephadex G-25M PD-10 gel-filtration column, Pharmacia), concentrated, and stored at -80 °C.

Preparation of Enriched Ferredoxin (Flavodoxin) NADPReductase

This was prepared from a biotin synthase plus cell-free extract with the Q Sepharose Fast Flow column (Fig.1). Fractions containing the enzyme were concentrated by ultrafiltration and stored at -80 °C.

Partial Purification of the Thiamine Pyrophosphate-dependent Protein

An enriched preparation, free from the other accessory proteins, was prepared from a biotin synthase plus cell-free extract by anion-exchange chromatography on Q Sepharose (Fig.1).

Assay Methods for the Biotin Synthase Accessory Proteins

Modifications of the assay described above were used to detect the accessory proteins during purification. All assays contained [^14C]dethiobiotin, cysteine, asparagine, and the cofactors described above, but Mg-ATP (250 nmol) and methionine (67 nmol) were used instead of S-adenosylmethionine. Using the enriched protein fractions, assays specific for each of the accessory proteins were developed. Flavodoxin activity was measured in a modified standard assay with enriched biotin synthase (50 µl) and the 0-45% ammonium sulfate fraction (80 µl). Ferredoxin (flavodoxin) NADP reductase was assayed using enriched biotin synthase containing the thiamine pyrophosphate-dependent protein (biotin synthase plus thiamine pyrophosphate, 50 µl) and enriched flavodoxin (50 µl) prepared by ammonium sulfate precipitation. The thiamine pyrophosphate-dependent protein was assayed using enriched biotin synthase (50 µl), pure flavodoxin (5 µl), and enriched ferredoxin (flavodoxin) NADP reductase (50 µl) prepared by anion-exchange chromatography.

The enriched fractions were largely undefined in terms of protein content, and the separation of biotin synthase and the accessory proteins was not always absolute. This resulted in weak background activity in some of the assays. For the purification of each protein, the fractions containing the strongest activity were selected for further steps. When the biotin synthase system was reconstituted using purer fractions, no background activity was detected in assays where one of the components was missed out.


RESULTS

Measurement of Biotin Synthase Activity in Vitro

Biotin synthase activity was measured in biotin synthase plus cell-free extracts by incubation with [^14C]dethiobiotin followed by isolation and analysis of the resulting [^14C]biotin. The addition of cofactors or metal ions was not necessary when freshly prepared extracts were used. [^14C]Biotin could be detected after 5-15 min of incubation. The reaction was not sensitive to oxygen. Fig.2shows the results of typical in vitro assays for biotin synthase with either [^14C]dethiobiotin or [S]cysteine as the source of radioactive label. Detection of [^14C]biotin was by TLC and autoradiography (Fig.2A) or by HPLC with on-line radiochemical detection (Fig.2B). When the cell-free extract was incubated with nonlabeled dethiobiotin plus [S]cysteine, the resulting biotin was S-labeled (Fig.2, A and C). For the experiments described below, the reaction conditions were controlled so that product formation was linear with respect to incubation time.


Figure 2: Measurement of biotin synthase activity in vitro. Panel A shows the results of a typical assay in vitrowith detection of the radiolabeled biotin by TLC and autoradiography. Lane1, [^14C]biotin; lane2, [^14C]dethiobiotin; lane 3, assay with [^14C]dethiobiotin; lane 4, assay with [S]cysteine. The assays in lanes3 and 4 were carried out using a biotin synthase plus cell-free extract (2.75 mg of protein/assay) and contained the standard cofactors and amino acids (see ``Materials and Methods''). B and C show the assays with [^14C]dethiobiotin and [S]cysteine analyzed by HPLC with on-line radiochemical detection. With [S]cysteine as the radioactive precursor, [S]biotin appears as a shoulder peak on a larger peak on the radioactivity trace. Estimation of [S]biotin production was therefore carried out with the Berthold Winflow radiochromatography software. More rigorous quantification would require further purification of the [S]biotin before HPLC analysis.



One of the radioactive compounds produced in the [^14C]dethiobiotin and [S]cysteine assays had the same chromatographic properties as a [^14C]biotin standard when analyzed using two TLC solvent systems and two HPLC methods (reverse-phase and paired-ion). Moreover, this compound was only detected in assays where both cell-free extract containing the cloned and over-expressed biotin synthase (bioB)gene and dethiobiotin were present, was derived from dethiobiotin ([^14C]dethiobiotin assay), and could be labeled with S ([S]cysteine assay). The compound also supported growth of the biotin auxotroph Lactobacillus plantarum. These results provide strong evidence that the compound was biotin. The identity of the other radioactive compounds is at present unknown.

Identification of the Low Molecular Weight Compounds Required for Biotin Synthase Activity in Vitro

The incorporation of S from cysteine into biotin by the biotin synthase plus cell-free extract suggested that cysteine was either the direct sulfur donor or closely related to the sulfur donor for the reaction. A cell-free extract, free of low molecular weight compounds, was prepared by gel filtration chromatography. When incubated with [^14C]dethiobiotin and cysteine or dethiobiotin and [S]cysteine, this extract had no biotin synthase activity. Activity could be restored by adding a low molecular weight fraction, which was prepared by passing the total cell-free extract through an ultrafiltration membrane with a molecular weight cut-off value of 10,000. The amount of biotin produced in this assay was increased by the addition of thiamine pyrophosphate, ATP, Fe, and a pyridine nucleotide. Since NADPH was the most effective of the pyridine nucleotides(17) , it was used in all subsequent experiments. However, alone or in combination these compounds only stimulated activity, and they could not substitute for the low molecular weight fraction. This showed that one or more compounds present in the low molecular weight fraction, in addition to those added to the assay, were required for activity.

These essential low molecular weight compounds could be provided by a protein-free fraction prepared from E. coli cells by boiling or ultrafiltration or by a solution of commercial yeast extract (Difco, 5 µl of a 100 mgbulletml solution in the assay). Initial attempts to isolate the active compounds either from the E. coli low molecular weight fraction or from yeast extract proved difficult but indicated that amino acids were involved. Consequently a screening approach was adopted in which amino acids were tested individually and in combinations. The results of these experiments (Table1) showed that the active compounds were methionine, cysteine, and one of asparagine, aspartate, glutamine, or serine. Methionine was required at a concentration of greater than 10 µM. Subsequent experiments showed that S-adenosylmethionine could substitute for ATP and methionine, indicating that S-adenosylmethionine is the active compound in vivo(31) . A concentration of cysteine of between 10 and 500 µM was required for activity, and D-cysteine could replace L-cysteine without any loss of activity. Asparagine, aspartate, glutamine, or serine at 1 mM resulted in very weak activity, whereas a concentration of 15 mM resulted in higher activity. Asparagine had the strongest effect, followed by aspartate, glutamine, and serine. The other 16 physiological amino acids (with the exception of cysteine and methionine, which have already been discussed) had no effect.



When cell-free systems were reconstituted from enriched and purified preparations of the proteins required for activity in vitro (see below), the following low molecular weight compounds, in addition to dethiobiotin, were essential for activity: S-adenosylmethionine; NADPH; Fe; cysteine; thiamine pyrophosphate; and one of the amino acids asparagine, aspartate, glutamine, or serine. This is the first report of the involvement of thiamine pyrophosphate and the amino acids asparagine, aspartate, glutamine, or serine with the biotin synthase system.

Evidence That Cysteine Is the Sulfur Donor for the Biotin Synthase Reaction

Potential sulfur donors for the biotin synthase reaction were tested in the assay system consisting of the desalted biotin synthase plus extract and the low molecular weight compounds required for activity. Addition of [S]cysteine or [S]cystine resulted in the production of [S]biotin. The incorporation of S from [S]cysteine into biotin in the presence of other sulfur compounds was then investigated (Table2). When concentrations of S-adenosylmethionine or homocysteine more than 11-fold in excess of the [S]cysteine concentration were added to the assay, the amount of S incorporated into biotin was unchanged, showing that the sulfur atoms from these compounds were not incorporated into biotin. However, when an excess of L-cysteine or D-cysteine was added, the amount of S incorporated into biotin was reduced to a level that was undetectable by HPLC analysis, showing that the labeled sulfur had been diluted out by the unlabeled sulfur.



If [S]methionine was used as the source of radiolabeled sulfur in the assay, an S-labeled compound with an R value similar to that of biotin was seen when the samples were analyzed by TLC and autoradiography (Fig.3). However, HPLC analysis showed that this compound was not biotin, and therefore methionine did not provide the sulfur atom for biotin. We have not yet identified this compound.


Figure 3: The sulfur atom from methionine is not incorporated into biotin in vitro. A desalted biotin synthase plus cell-free extract was incubated in a standard assay with [S]methionine (10 µCi, 10 pmol) as the source of radioactive label. The resulting radiolabeled compounds were analyzed by TLC and autoradiography and by HPLC with on-line radiochemical detection. A, analysis by TLC showed a S-labeled compound with the same R value as biotin (arrow) in addition to several other radiolabeled compounds. B, however, analysis by reverse-phase HPLC showed that this compound was not biotin. The arrow indicates the elution position of biotin.



If S-adenosylmethionine was the sulfur donor, S from labeled methionine would be incorporated into biotin, as methionine is a precursor for S-adenosylmethionine. This provides further evidence that S-adenosylmethionine is not the sulfur donor for biotin synthesis in E. coli.

Requirement by Biotin Synthase for Accessory Proteins

The proteins in a biotin synthase plus cell-free extract were separated on an anion-exchange column as indicated in Fig.1, and fractions were assayed for biotin synthase activity. No activity was found in any of the fractions. However, when a biotin synthase minus cell-free extract, which alone had no detectable biotin synthase activity in vitro, was added to the fractions, a clear peak of biotin synthase activity was detected. This showed that one or more proteins in addition to biotin synthase were required for activity and that the loss of biotin synthase activity after the anion-exchange column was not due to inactivation of the biotin synthase itself but due to the absence of the accessory protein or proteins.

Investigation of the Proteins Required for Biotin Synthase Activity in Vitro

Having established that one or more proteins in addition to biotin synthase were essential for activity in vitro, we developed assays specific for these proteins. These assays contained biotin synthase (free of the accessory proteins and prepared by anion-exchange chromatography, as described under ``Materials and Methods''), low molecular weight components, and the fractions to be tested.

Fractionation of a biotin synthase minus cell-free extract by ammonium sulfate precipitation led to the identification of two active fractions: the proteins precipitating between 0 and 45% ammonium sulfate saturation, and those remaining in the supernatant after 55% saturation. Both of these fractions were required to restore activity to the biotin synthase preparation.

Purification and Identification of the Active Protein in the 55%+ Ammonium Sulfate Fraction

The desalted supernatant from the 55% ammonium sulfate precipitation step was loaded onto a Q Sepharose Fast Flow (Pharmacia) column as described for the preparation of enriched biotin synthase. The active component was eluted with a linear NaCl gradient, 0-600 mM in 1500 ml (Fig.1). Active fractions were pooled and further purified by a second anion-exchange step (HiLoad Q Sepharose High Performance 26/10, Pharmacia) using the same buffer system (20 mM Tris, pH 7.5 containing 1 mM dithiothreitol). The active protein was eluted with a linear NaCl gradient (0-1 M in 2 liters). The active fractions from this step were pooled and concentrated by ultrafiltration (YM10 membrane, Amicon) before loading onto a gel-filtration column (Sephacryl HR-100, 16 mm 500 mm column) equilibrated with 50 mM Tris buffer, pH 7.5, containing 1 mM dithiothreitol. The last step in the purification was anion-exchange chromatography on a Mono Q 5/5 column (Pharmacia) using the Tris/dithiothreitol buffer system at pH 7 and elution with a NaCl gradient.

In the later stages of purification, a strong yellow color was associated with the active fractions. After the Mono Q step, SDS-PAGE of the active fractions showed one fraction to contain one major protein band with a M(r) of 19,000 (Fig.4A). An absorption spectrum of the purified active protein showed a typical flavoprotein spectrum (Fig.4B).


Figure 4: SDS-PAGE and absorbance spectrum of purified flavodoxin. A, SDS-PAGE was carried out on a 15% gel that was stained with Coomasie Blue; B, the absorption spectrum was recorded on a Ciba-Corning Spectrascan 2800 spectrophotometer.



The protein shown in Fig.4A was digested with trypsin, and, from the resulting 17 peptide fractions purified by HPLC, two were sequenced. The amino acid sequences were Ala-Ile-ThrGly-Ile-Phe-Phe-Gly-Ser-Asp-Thr-Gly-Asn-Thr-Glu-Asn-Ile-AlaLys and Gly-Leu-Ala-Asp-Asp-Asp-His-Phe-Val-Gly-Leu-Ala-Ile-Asp-Glu-Asp-Arg, which correspond to residues 1-19 and 130-147, respectively, of E. coli flavodoxin. The minor band seen on SDS-PAGE was presumably the flavodoxin apoprotein that is often seen in preparations of this protein(32, 33) .

The Two Accessory Proteins in the 0-45% Ammonium Sulfate Fraction

Two protein fractions from the Q Sepharose anion-exchange separation of the biotin synthase plus extract shown in Fig.1were able, when combined, to substitute for the 0-45% ammonium sulfate fraction from the biotin synthase minus extract. We therefore used these fractions for the purification of the proteins. One of the proteins has been purified to homogeneity and identified as ferredoxin (flavodoxin) NADP reductase (details below). The second protein eluted from the Q Sepharose column shown in Fig.1at a NaCl concentration of approximately 0.5 M and required thiamine pyrophosphate for stabilization of activity during purification. We will present details of its purification and identity as soon as possible.

Purification and Identification of Ferredoxin (Flavodoxin) NADPReductase

A 100,000 g supernatant from the biotin synthase plus cell-free extract was loaded onto a Pharmacia Q Sepharose Fast Flow anion-exchange column. Active fractions (Fig.1) were pooled and concentrated by ultrafiltration (YM10 membrane, Amicon). The active component was then processed through Mono Q anion-exchange (at different pH values and with different NaCl gradients, three stages of the purification), Superose 12 Prep gel-filtration, and Sephacryl HR 100 gel-filtration chromatography columns until an active fraction containing one major protein band with a M(r) of about 29,000 as judged by SDS-PAGE was obtained (Fig.5). The protein was cut from a Western blot and sequenced directly. The N-terminal sequence obtained was Met-Ala-Asp-Trp-Val-Thr-Gly-Lys-Val-Thr-Lys-Val-Gln-Asn-Trp, which corresponds to the N-terminal sequence of E. coli ferredoxin (flavodoxin) NADP reductase (EC 1.18.1.2)(34) . This information agreed with our knowledge of the involvement of flavodoxin in the biotin synthase system. A more detailed account of the purification procedure used in order to obtain a partial amino acid sequence for this protein is not given since, following its identification, it is clear that more elegant purification methods exist in the literature(35) .


Figure 5: SDS-PAGE of purified ferredoxin (flavodoxin) NADP reductase. Samples were analyzed with a silver-stained 10-15% gradient gel (Pharmacia Phast System). The two minor bands were not visible on a Coomasie Blue-stained SDS gel, or following Western blotting and staining with Amido Black.



Reconstitution of Active Biotin Synthase Systems in Vitro

Active biotin synthase systems were reconstituted in vitro using the enriched preparations of biotin synthase and the thiamine pyrophosphate-dependent protein and either pure flavodoxin plus an enriched ferredoxin (flavodoxin) NADP reductase preparation or pure ferredoxin (flavodoxin) NADP reductase plus an enriched flavodoxin preparation. All of the low molecular weight factors identified above, namely thiamine pyrophosphate; Fe; NADPH; S-adenosylmethionine; and one of the amino acids asparagine, aspartate, glutamine, or serine were essential in these systems. KCl, which was shown to stimulate biotin synthase in a cell-free extract of E. coli(15) had no effect and could not substitute for asparagine, aspartate, glutamine, or serine.


DISCUSSION

We have developed sensitive radiochemical methods for the measurement of biotin synthase activity in vitro and have used them to further our knowledge of the biotin synthase reaction. The keys to developing this assay were the cloning of the bioB (biotin synthase) gene, the preparation of a concentrated cell-free extract (up to 100 mgbulletml protein concentration), the use of radioactively labeled precursors ([^14C]dethiobiotin or [S]cysteine/cystine), postreaction concentration and purification of the resulting radioactive biotin by solid phase extraction and careful attention to methods of analysis and detection (TLC and HPLC). This assay system has been used to identify the low molecular weight factors and two of the proteins that are required for biotin synthase activity in vitro.

The method commonly used for measuring biotin synthesis in vitro is based on the growth of microorganisms auxotrophic for biotin, the most widely used microorganism being L. plantarum(36) . Although the method is sensitive (0.1 ng or 0.41 pmol of biotin), it is slow and labor intensive. It is also subject to disturbances that can make quantification difficult and requires many controls to be carried out. In addition, it has been shown that Lactobacillus plantarum can grow on concentrations of dethiobiotin (the substrate for biotin synthase) greater than 1 µM(37) . Concentrations of 5 µM are commonly used in assays. Other compounds being tested in the assays may also affect the growth of the test organism.

The radiochemical assays that we have introduced are sensitive and specific, and can easily be automated. With regard to sensitivity, we routinely measure the production of 24.4 ng or 100 pmol of biotin in our standard assays with [^14C]dethiobiotin as substrate, and we have measured the production of sub pmol amounts of biotin with [S]cysteine as substrate. The assays can be used semiquantitatively with TLC and x-ray film to quickly identify active fractions from a chromatography column or can be quantified by HPLC with on-line radiochemical detection, radiochemical scanning of TLC plates, or densitometry of the resulting x-ray film. The radiochemical assays are therefore better suited for assaying large numbers of fractions and for kinetic experiments.

Cysteine is taken up by E. coli as cystine and reduced to cysteine in the cell(21, 22) . The path of sulfur into biotin from cysteine could then be as follows: (a) direct involvement in the biotin synthase reaction, in which case cysteine is the direct sulfur donor; (b) transfer of the sulfur from cysteine to a protein that would then transfer the sulfur to the biotin synthase reaction. Here the protein would be the sulfur donor, and cysteine would be the final metabolic intermediate or low molecular weight sulfur donor; (c) transfer of the sulfur from cysteine to a low molecular weight compound that would then act as the sulfur donor; or (d) conversion of cysteine itself to the direct sulfur donor in one or more steps. The results of DeMoll and Shive(21, 22) showed that the origin of the sulfur atom in biotin in E. coli was cysteine, but because of the nature of their experiments, they were not able to discriminate between the possibilities described above. Groups working both with E. coli and other microorganisms have proposed methionine and S-adenosylmethionine (15) as possible sulfur donors. Our investigations have been carried out in a defined system in vitro, containing only known low molecular weight compounds. Our results confirmed that cysteine is the origin of the sulfur atom in biotin and ruled out methionine, S-adenosylmethionine, and homocysteine as sulfur donors. In addition, our results rule out the possibility that the sulfur atom is transferred to an unknown low molecular weight compound before involvement in the biotin synthase reaction (possibility c above). In bacteria, the known metabolic fates of cysteine are degradation to pyruvate and H(2)S or conversion to methionine via homocysteine. We have shown that homocysteine is not the sulfur donor for the reaction. The possibilities remain that cysteine is converted to the direct sulfur donor by an unknown pathway or that the sulfur is transferred to an unknown protein before incorporation into biotin. Our recent results (^2)rule out both of these possibilities. One intriguing result of our investigations was that D-cysteine could replace L-cysteine in the assays. Whether an amino acid racemase or cysteine-specific racemase is present in the cell-free extract is not yet clear. Other possibilities are that the cysteine sulfur is transferred to the reaction by an enzyme not specific for a particular isomer or even nonenzymatically.

The role of thiamine pyrophosphate in the biotin synthase system is not yet clear, although we know that it is required to maintain the activity of one of the proteins in the 0-45% ammonium sulfate fraction. It is difficult to envisage a thiamine pyrophosphate-dependent enzyme being involved directly in the biotin synthase reaction, since thiamine pyrophosphate is a cofactor for enzymes that cleave or form carbon-carbon bonds next to carbonyl groups(38) .

The role of the amino acids asparagine, aspartate, glutamine, and serine, which we have shown to be essential for the reaction, is also unknown, but we hope that identification of all of the reaction components will lead to the clarification of their function. For optimal activity in the assays in vitro, the amino acids are required at concentrations above those found naturally in E. coli cells and they may be directly involved in the reaction in vitro, or may have a regulatory role. KCl, which has been reported to have a stimulatory effect on biotin synthesis in vitro(15) , was unable to substitute for these amino acids, so it is unlikely that the effect is solely ionic.

The requirement of the biotin synthase system for S-adenosylmethionine, NADPH, and Fe together with flavodoxin and ferredoxin (flavodoxin) NADP reductase, suggests similarities between biotin synthase and other E. coli enzymes. Both pyruvate formate-lyase (39, 40) and anaerobic ribonucleotide reductase from E. coli(41) require S-adenosylmethionine for activation. In these systems, the reductive cleavage of Sadenosylmethionine generates an enzyme-bound radical that both activates the enzyme and is essential for the reaction. These similarities between biotin synthase, ribonucleotide reductase, and pyruvate formate-lyase provide further evidence that biotin synthesis may proceed by a radical mechanism, as proposed in (19) . A mechanism could be envisaged in which an electron from NADPH is supplied via flavodoxin for the reductive cleavage of S-adenosylmethionine with the concomitant generation of a radical on biotin synthase, which would then abstract a hydrogen radical from the methyl or methylene carbon of dethiobiotin. Sulfur from the sulfur donor would then become covalently bound either to the methyl or methylene radical of dethiobiotin, whichever becomes activated first, and then the second carbon to be linked to sulfur would be activated in a similar way. Overall, the introduction of sulfur between the carbon atoms of dethiobiotin to form biotin would involve sequential activation of each of those carbons by radical formation, and each activation would involve the provision of one electron by flavodoxin and the cleavage of one molecule of S-adenosylmethionine, that is, a total of two electrons and two molecules of S-adenosylmethionine/molecule of biotin formed. We are currently investigating this proposal. This model for the reaction takes into account several factors that we have shown to be involved in the biotin synthase reaction such as flavodoxin, ferredoxin (flavodoxin) NADP reductase, NADPH, Fe, and S-adenosylmethionine. However, it does not account for additional protein and low molecular weight factors that are also required for the reaction in vitro.

Both pyruvate formate-lyase and anaerobic ribonucleotide reductase are extremely sensitive to oxygen and are rapidly inactivated under aerobic conditions. This is not the case for biotin synthase ( (42) and this paper), suggesting some differences between the mechanism of biotin synthase and the enzymes described above. Only the complete purification of all the components of the biotin synthase system from E. coli will clarify this point. To our knowledge, our results are the first indication that S-adenosylmethionine may be involved in the generation of radicals in aerobic enzyme systems.

Enzymes that proceed by a radical mechanism usually have a requirement for a metal ion(43) . Recent work where biotin synthase was purified (42) and our results^2 provide evidence that the enzyme has a [2Fe-2S] center. The enzyme system also requires free iron for activity(15, 16, 17) , and our results indicate that this is required for the binding of cysteine to biotin synthase.^2

The biotin synthases from E. coli, B. sphaericus, and Saccharomyces cerevisiae show extensive sequence similarity(44) . It seems likely therefore that the mechanisms of these enzymes, and those from other bacteria, for example Serratia marcescens(45) , will be similar and that they will also use the low molecular weight compounds and the accessory proteins described in this paper. However, the involvement of FAD in the B. sphaericus enzyme system (23) suggests that there may also be some differences between the enzymes from different organisms.

The reaction catalyzed by the bioB gene product, biotin synthase, does not require ATP, and therefore the enzyme should be named biotin synthase and not biotin synthetase. In view of the multicomponent nature of the enzyme in vitro, we suggest the names biotin synthase for the bioB gene product (on genetic grounds, since no other specific mutations affecting the last step in the biotin pathway in E. coli have been found) and biotin synthase system for biotin synthase plus all the cofactors and proteins necessary for activity in vitro.

To summarize, we have developed a radiochemical assay for the measurement of biotin synthase activity and used it to identify all of the low molecular weight compounds and two of the proteins required for activity in vitro. A third essential protein is currently being purified.

Since we first reported the involvement of flavodoxin, ferredoxin (flavodoxin) NADP reductase, and a third thiamine pyrophosphate dependent protein in the E. coli biotin synthase system(17) , the involvement of flavodoxin has been confirmed (46) .


FOOTNOTES

*
The major findings in this paper are contained in Patent Cooperation Treaty Patent Application WO 94/08023. The microbiological and fermentation contributions to the work described in the patent application were carried out in the laboratory of Dr. Johannes Brass. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 41-28-48-59-37; Fax: 41-28-48-61-80.

^1
The abbreviations used are: TLC, thin layer chromatography; HPLC, high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis.

^2
O. M. Birch and N. M. Shaw, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank Bruno Lehner and Jean-François Burkhalter for expert technical assistance, Drs. Séverin Frutiger and Graham Hughes (Centre Medical Universitaire, Geneva) for amino acid sequencing, and Professor J. E. Baldwin (Oxford) for helpful discussions concerning possible reaction mechanisms for the biotin synthase reaction.


REFERENCES

  1. Eisenberg, M. (1987) in Escherichia coli and Salmonella typhimurium (Neidhardt, F. C., ed) Vol. 1, pp. 544-550, American Society for Microbiology, Washington, D. C.
  2. Baldet, P., Gerbling, H., Axiotis, S., and Douce, R. (1993) Eur. J. Biochem. 217,479-485 [Abstract]
  3. Knowles, J. R. (1989) Annu. Rev. Biochem. 58,195-221 [CrossRef][Medline] [Order article via Infotrieve]
  4. Goldberg, M. W., and Sternbach, L. H. (Nov. 22, 1949) U. S. Patents 2,489,232, 2,489,235, and 2,489,238
  5. McGarrity, J., and Tenud, L. H. (Oct. 24, 1989) U. S. Patent 4,876,350
  6. Brown, S. W., and Kamogawa, K. (1991) Biotechnol. Genet. Eng. Rev. 9,295-326 [Medline] [Order article via Infotrieve]
  7. Lévy-Schil, S., Debussche, L., Rigault, S., Soubrier, F., Bacchetta, F., Lagneaux, D., Schleuniger, J., Blanche, F., Crouzet, J., and Mayaux, J.-F. (1993) Appl. Microbiol. Biotech. 38,755-762
  8. Ohsawa, I., Kisou, T., Kodama, K., Yoneda, I., Speck, D., Gloeckler, R., Lemoine, Y., and Kamogawa, K. (1992) J. Ferment. Bioeng. 73,121-124
  9. Sanyal, I., Lee, S.-L., and Flint, D. (1994) J. Am. Chem. Soc. 116,2637-2638
  10. Ifuku, O., Miyaoka, H., Koga, N., Kishimoto, J., Haze, S., Wachi, Y., and Kajiwara, M. (1994) Eur. J. Biochem. 220,585-591 [Abstract]
  11. Rolfe, B., and Eisenberg, M. A. (1968) J. Bacteriol. 96,515-524 [Medline] [Order article via Infotrieve]
  12. Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., and Lipscomb, J. D. (1989) J. Biol. Chem. 264,21677-21681 [Abstract/Free Full Text]
  13. Reed, K. E., and Cronan, J. E. (1993) J. Bacteriol. 175,1325-1336 [Abstract]
  14. DeMoll, E., and Shive, W. (1985) Biochem. Biophys. Res. Commun. 132,217-222 [Medline] [Order article via Infotrieve]
  15. Ifuku, O., Kishimoto, J., Haze, S., Yanagi, M., and Fukushima, S. (1992) Biosci. Biotechnol. Biochem. 56,1780-1785 [Medline] [Order article via Infotrieve]
  16. Fujisawa, A., Abe, T., Ohsawa, I., Kamogawa, K., and Izumi, Y. (1993) FEMS Microbiol. Lett. 110,1-4
  17. Birch, O. M., Brass, J., Fuhrmann, M., and Shaw, N. M. (Apr. 14, 1994) PCT Patent Application WO 94/08023
  18. Baxter, R. L., Camp, D. J., Coutts, A., and Shaw, N. (1992) J. Chem. Soc. Perkin Trans. 1,255-258
  19. Marti, F. B. (1983) Eidgenossischen Technischen Hochschule, Doctoral dissertation 7236, ETH Zurich
  20. Marquet, A., Frappier, F., Guillerm, G., Azoulay, M., Florentin, D., and Tabet, J.-C. (1993) J. Am. Chem. Soc. 115,2193-2145
  21. DeMoll, E., and Shive, W. (1983) Biochem. Biophys. Res. Commun. 110,243-249 [Medline] [Order article via Infotrieve]
  22. DeMoll, E., White, R. H., and Shive, W. (1984) Biochemistry 23,558-562 [Medline] [Order article via Infotrieve]
  23. Florentin, D., Tse Sum Bui, B., Marquet, A., Oshiro, T., and Izumi, Y. (1994) C. R. Acad. Sci. Paris 317,485-488 [Medline] [Order article via Infotrieve]
  24. Barker, D. F., and Campbell, A. M. (1980) J. Bacteriol. 143,789-800 [Medline] [Order article via Infotrieve]
  25. Bullock, W. O., Fernandez, J. M., and Short, J. M. (1987) BioTechniques 5,376-379
  26. Hughes, G. J., Frutiger, S., Paquet, N., and Jaton, J.-C. (1990) Biochem. J. 271,641-647 [Medline] [Order article via Infotrieve]
  27. Ferreira, R. B., and Davies, D. D. (1987) Plant Physiol. 83,869-877
  28. Schägger, H., and Von Jagow, G. (1987) Anal. Biochem. 166,368-379 [Medline] [Order article via Infotrieve]
  29. Matsudaira, P. (1987) J. Biol. Chem. 262,10035-10038 [Abstract/Free Full Text]
  30. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 76,4350-4354 [Abstract]
  31. Markham, G. D., Hafner, E. W., Tabor, C. W., and Tabor, H. (1980) J. Biol. Chem. 255,9082-9092 [Abstract/Free Full Text]
  32. Osborne, C., Chen, L.-M., and Matthews, R. G. (1991) J. Bacteriol. 173,1729-1737 [Medline] [Order article via Infotrieve]
  33. Mayhew, S. G., and Tollin, G. (1992) Chem. Biochem. Flavoenzymes 3,389-426
  34. Bianchi, V., Reichard, P., Eliasson, R., Pontis, E., Krook, M., Jörnvall, H., and Haggard-Ljungquist, E. (1993) J. Bacteriol. 175,1590-1595 [Abstract]
  35. Eliasson, R., Pontis, E., Fontecave, M., Gerez, C., Harder, J., Jörnvall, H., Krook, M., and Reichard, P. (1992) J. Biol. Chem. 267,25541-25547 [Abstract/Free Full Text]
  36. Gaudry, M., and Ploux, O. (1992) Chromatogr. Sci. J. 60,441-467
  37. Bowman, W. C., and DeMoll, E. (1993) J. Bacteriol. 175,7702-7704 [Abstract]
  38. Friedrich, W. (1987) Handbuch der Vitamine, pp. 220-254, Urban and Schwarzenberg, M ü nchen, Germany
  39. Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,1332-1335 [Abstract]
  40. Wagner, A. F. V., Frey, M., Neugebauer, F. A., Schäfer, W., and Knappe, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,996-1000 [Abstract]
  41. Reichard, P. (1993) J. Biol. Chem. 268,8383-8386 [Free Full Text]
  42. Sanyal, I., Cohen, G., and Flint, D. H. (1994) Biochemistry 33,3625-3631 [Medline] [Order article via Infotrieve]
  43. Stubbe, J. (1989) Annu. Rev. Biochem. 58,257-285 [CrossRef][Medline] [Order article via Infotrieve]
  44. Zhang, S., Sanyal, I., Bulboaca, H., Rich, A., and Flint, D. (1994) Arch. Biochem. Biophys. 309,29-35 [CrossRef][Medline] [Order article via Infotrieve]
  45. Sakurai, N., Imai, Y., Komatsubara, S., and Tosa, T. (1994) J. Ferment. Bioeng. 77,610-616
  46. Ifuku, O., Koga, N., Haze, S., Kishimoto, J., and Wachi, Y. (1994) Eur. J. Biochem. 224,173-178 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.