(Received for publication, January 9, 1995; and in revised form, May 9, 1995)
From the
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
[C]dethiobiotin, which was converted to
[
C]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.
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.
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 (-max, Amersham Corp.) for autoradiography.
Samples for quantitative analysis were resuspended in 50 mM NaHPO
, pH 3.5, containing 20 mM triethylamine and 203 mg
liter
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
PO
, 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
ml
min
and the total flow through the cell was
1.75 ml
min
, 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 KHPO
, pH 6.5, plus 4.2 mM tetrabutyl ammonium phosphate and 16% (v/v) methanol. The column
was at 40 °C.
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
mg
ml
of protein with 20 mM Tris
buffer, pH 7.5, containing 1 mM dithiothreitol and 1
g
liter
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 ml
min
, 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
--
line shows the
NaCl gradient.
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.
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,
[C]biotin; lane2,
[
C]dethiobiotin; lane 3, assay with
[
C]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
[
C]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
[C]dethiobiotin and
[
S]cysteine assays had the same chromatographic
properties as a [
C]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 ([
C]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.
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 mgml
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.
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.
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.
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 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) .
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.
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 mgml
protein concentration), the use of
radioactively labeled precursors
([
C]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
[C]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
HS 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 (
)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 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.
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) .