From the Department of Biochemistry, University College Dublin,
Belfield, Dublin 4, Ireland
The genes that encode the two different subunits
of the novel electron-transferring flavoprotein (ETF) from
Megasphaera elsdenii were identified by screening a partial
genomic DNA library with a probe that was generated by amplification of
genomic sequences using the polymerase chain reaction. The cloned genes
are arranged in tandem with the coding sequence for the
-subunit in
the position 5' to the
-subunit coding sequence. Amino acid sequence
analysis of the two subunits revealed that there are two possible
dinucleotide-binding sites on the
-subunit and one on the
-subunit. Comparison of M. elsdenii ETF amino acid
sequence to other ETFs and ETF-like proteins indicates that while
homology occurs with the mitochondrial ETF and bacterial ETFs, the
greatest similarity is with the putative ETFs from clostridia and with
fixAB gene products from nitrogen-fixing bacteria. The
recombinant ETF was isolated from extracts of Escherichia coli. It is a heterodimer with subunits identical in size to the native protein. The isolated enzyme contains approximately 1 mol of
FAD, but like the native protein it binds additional flavin to give a
total of about 2 mol of FAD/dimer. It serves as an electron donor to
butyryl-CoA dehydrogenase, and it also has NADH dehydrogenase activity.
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INTRODUCTION |
Electron-transferring flavoproteins
(ETFs)1 catalyze electron
transfer between other flavoproteins (1). The pig liver enzyme is the
best characterized ETF. It is found in the mitochondrion, where it
functions to transfer electrons from flavoprotein dehydrogenases involved in the metabolism of fatty acids, choline, and amino acids to
a further flavoprotein, named ETF-CoQ oxidoreductase. The latter enzyme
passes the electrons to the terminal electron transfer chain (1).
Mammalian ETF is a heterodimer that contains one FAD and one AMP (2,
3). The FAD is the redox center in the protein, while the AMP is
thought to have only a structural role. The genes that encode the human
enzyme have been cloned (4, 5), and the three-dimensional structure of
the recombinant protein shows that the FAD interacts mainly with the
-subunit, while the AMP is buried in the
-subunit (6). A
deficiency in the human enzyme causes the disease glutaric aciduria
type II, an often fatal inborn error of fatty acid and amino acid
metabolism, emphasizing the importance of this protein (7).
Proteins similar to mammalian ETF have been isolated from bacteria.
ETFs from Paracoccus denitrificans and the methylotroph W3A1
are also heterodimers with one FAD and one AMP per molecule (3, 8-10).
The mammalian and P. denitrificans enzymes share very
similar catalytic properties (11). Although the physicochemical properties of methylotroph W3A1 ETF are similar to those of the other
ETFs, the methylotroph protein is not able to replace the other ETFs in
catalytic assays (10, 12). In both organisms, the genes are arranged in
tandem with the gene for the
-subunit preceding the
-subunit gene
(13, 14). Sequence analysis identified similar genes in a variety of
other organisms. Thus, the proteins encoded by the fixB and
fixA genes of nitrogen-fixing bacteria have similar
sequences to the
- and
-subunits, respectively, of ETF, and their
genetic organization is similar to that of bacterial ETF (5, 13-15).
Although there is evidence that the proteins encoded by the
fixAB genes are involved in nitrogen fixation, their precise
function has not been established (16, 17).
The ETF in the anaerobic bacterium Megasphaera elsdenii
functions in a similar way to the ETFs described above, but it differs in several of its physicochemical and catalytic properties. The organism is found in the rumen of sheep and cattle, where it ferments lactate (18), disposing of excess reducing equivalents either by
forming molecular hydrogen or through the production of short-chain fatty acids (19-21). The ETF mediates electron transfer from the flavoprotein D-lactate dehydrogenase to a third
flavoprotein, termed butyryl-CoA dehydrogenase (BCD), that functions
physiologically by reducing enoyl-CoA to acyl-CoA (21-24). M. elsdenii ETF has two subunits of different molecular mass, and it
contains FAD, which acts as its sole redox group (24). However, in
contrast to the ETF preparations from other sources described above,
M. elsdenii ETF contains approximately 1.4 mol of FAD in the
form in which it is isolated, and it binds additional FAD so that when saturated with flavin it has 2 mol of FAD/dimer (24). A variable fraction of the flavin in this ETF comprises two hydroxylated derivatives of FAD: 6-OH-FAD
(6-hydroxy-7,8-dimethyl-10(5'-ADP-ribityl)-isoalloxazine) and 8-OH-FAD
(7-methyl-8-hydroxy-10(5'-ADP-ribityl)-isoalloxazine) (24-29). The
hydroxyflavins usually occur in small amounts, but at the highest
levels observed they make up about half the flavin in the enzyme. There
is evidence that they are artifacts generated during isolation of this
ETF (22). Their optical properties differ from those of FAD, and they
also affect the catalytic properties of the enzyme. They have not been
observed in preparations of ETF from other sources. In further contrast
to other ETFs, the M. elsdenii enzyme does not contain AMP
(26). In addition, it catalyzes the oxidation of NADH, thus allowing
electron transfer from NADH to BCD and to other electron acceptors
including 2,6-dichlorophenolindophenol (DCPIP) (22, 24).
Incubation of the apoenzyme of M. elsdenii ETF with
8-halogenated FAD derivatives led to covalent attachment of flavin to the
-subunit, suggesting that the flavin sites are on this subunit (30, 31). The analogue-substituted ETF is reducible by NADH, and it
retains diaphorase activity, but it is inactive with BCD. Similar
experiments using triazine dyes as affinity labels led to the
conclusion that the NADH binding site is also on the
-subunit (32).
M. elsdenii ETF stabilizes the red anionic form of the flavin semiquinone during reductive titration with dithionite ion, as
also occurs with ETFs from other sources (1, 24). However, semiquinone
is not observed during electrochemical reduction of the enzyme in the
presence of mediator dyes, leading to the conclusion that stabilization
of the semiquinone is kinetic rather than thermodynamic (33). The
midpoint potential for the overall two-electron reduction of the enzyme
is
0.259 V.
The properties of M. elsdenii ETF show that although overall
it resembles ETF preparations from other sources, it is unique in
binding two molecules of FAD, in lacking AMP, and in catalyzing NADH
oxidation. With the aim of using structural analysis to further elucidate the similarities with and differences from other ETFs, we
have cloned the genes encoding the M. elsdenii protein. The base sequences of the genes and the predicted amino acid sequences of
the subunits have been compared with those of other ETFs. Purified recombinant protein has been produced using an Escherichia
coli expression system, and the properties of the recombinant
protein have been compared with those of native ETF. These studies lay the foundation for more detailed structural analysis by x-ray crystallography (34) and site-directed mutagenesis. A preliminary report of some of the findings has been published (35).
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EXPERIMENTAL PROCEDURES |
Growth of Bacteria--
Cells of M. elsdenii (strain
LC1, NCIMB 8927) were prepared as described previously (24).
Escherichia coli strains TG1 and DH5
were maintained and
propagated aerobically in Luria-Bertani medium (36). Anaerobic cultures
of E. coli were also grown in Luria-Bertani medium using the
techniques described for M. elsdenii. A large scale culture
of E. coli (40 liters) was grown under anaerobic conditions
to obtain sufficient quantities of the recombinant protein for
biochemical characterization. The growth medium (1% (w/v) tryptone,
0.5% (w/v) yeast extract, 0.5% (w/v) NaCl, 0.5% (w/v) glucose, 17 mM KH2PO4, 72 mM
K2HPO4) was supplemented with 200 µg/ml
ampicillin, and the culture was induced by adding IPTG to a final
concentration of 0.5 mM in early log phase when
A590 = 0.13.
Protein Purification--
Native ETF was purified from M. elsdenii as described previously (24). Recombinant M. elsdenii ETF was purified from E. coli transformed with
pBETFfl.1 as follows. All procedures were carried out at 4 °C. The
buffers contained 0.3 mM EDTA, and they were at pH 6 unless
otherwise noted. A crude extract of E. coli was made by
suspending cell paste in 20 mM potassium phosphate buffer,
pH 7.0 (3 ml/g); lysing the cells by sonication (Branson sonicator,
type 7532B operated at 80% full power); and centrifuging (23,500 × g for 20 min). The extract (350 ml) was applied to a column of Q-Sepharose (42 × 2.1-cm diameter) equilibrated with 50 mM potassium phosphate buffer. The column was washed with
0.1 M potassium phosphate buffer (460 ml), and the ETF
eluted using a linear salt gradient made by continuously diluting 500 ml of 0.1 M potassium phosphate buffer with 500 ml of 0.33 M potassium phosphate buffer. Fractions that contained
diaphorase activity were pooled, diluted to 50 mM potassium
phosphate, and applied to a second Q-Sepharose column (37.5 × 1.6-cm diameter). It was washed with 300 ml of 0.1 M
potassium phosphate buffer and eluted with the gradient used earlier
except for the total volume of buffer in the gradient, which was 600 ml. Fractions with an absorbance ratio
(A272:A450) less than 10 were pooled. Solid ammonium sulfate was added to a concentration of 1 M. The solution was then applied to a column of
phenyl-Sepharose CL-4B (44 × 2.1-cm diameter) equilibrated with
50 mM potassium phosphate and 1 M ammonium
sulfate. A yellow band with bright yellow-green fluorescence was bound
tightly to the top of the column. The column was washed with 600 ml of
the same buffer, and the ETF was eluted with a linear gradient in which
500 ml of 1 M ammonium sulfate in 50 mM
potassium phosphate buffer was continuously diluted with 500 ml of 50%
(v/v) ethylene glycol in the same buffer. Fractions were analyzed by
SDS-PAGE. Those that gave only two bands corresponding to the two
subunits of ETF were pooled, concentrated by ultrafiltration using a
PM30 (Amicon Corp.) membrane, dialyzed versus 0.1 M potassium phosphate, and stored at
20 °C.
Enzyme Assays--
ETF activity was measured by coupling the
oxidation of NADH to the reduction of crotonyl-CoA in the presence of
butyryl-CoA dehydrogenase (21, 24). The assay contained in a final
volume of 1 ml at 25 °C 30 µM crotonyl-CoA, 60 mM potassium phosphate, pH 6, 0.1 mM NADH, 2.3 µM BCD, and ETF. One unit of ETF activity in this assay
is defined as an absorbance change of 1 per min at 340 nm.
Diaphorase activity was measured by coupling the oxidation of NADH to
the reduction of DCPIP. The reaction mixture contained 0.14 mM NADH, 32 µM DCPIP, 0.1 M
potassium phosphate buffer, pH 7.0, and ETF in a final volume of 1 ml
at 25 °C. The decrease in absorbance at 600 nm was measured. One
unit of diaphorase activity is defined as an absorbance change of 1 per
min.
Analytical Methods--
Protein was determined (37) using bovine
serum albumin as a standard and a Biuret coefficient of 0.833 (24).
The flavin chromophore was extracted from recombinant ETF by heat
treatment in a sealed 1.5-ml centrifuge tube at 90 °C (27). The
precipitated protein was removed centrifugation (20,500 × g, 10 min).
The enzyme was prepared for N-terminal sequencing by first using
SDS-PAGE to separate the subunits. The areas of the gel that contained
the two subunits were then excised separately, and the two proteins
electroeluted (40). They were precipitated with 12.5% (w/v)
trichloroacetic acid. N-terminal amino acid sequencing was carried out
by automated Edman degradation using an Applied Biosystems 4778 protein
sequencer.
PAGE and SDS-PAGE analyses were performed (39) using a vertical slab
gel (ATTO AE-6450). The separated proteins were stained with Coomassie
Blue or for NADH dehydrogenase activity as described later.
DNA Manipulations--
Cloning and transformation techniques
were carried out essentially as described (36). Plasmid DNA was
isolated by the alkaline lysis method (36). M. elsdenii
genomic DNA was isolated and purified using the procedure described
(40). Approximately 2.5 mg of DNA was obtained from 0.12 g of cell
paste when the culture was harvested after 10-12 h of growth.
The polymerase chain reaction (PCR) was used to amplify genomic DNA
sequences. Reactions were carried out in a reaction volume of 100 µl
that contained 0.1 µg of genomic DNA; a 1 µM
concentration each of the primers A and B as given below; 2.0 mM MgCl2; 50 mM KCl; 10 mM Tris-HCl buffer, pH 9.0, 25 °C; 0.1% (w/v)
Triton® X-100; a 0.25 mM concentration each of
the four dNTPs; 5 units of Taq polymerase (Promega Corp.).
PCR amplifications were carried out in a Techne thermocycler. The
conditions were as follows: 30 cycles of denaturation, 94 °C for 1.5 min; annealing, 40 °C for 1 min; extension, 72 °C for 3 min.
Primer A is nondegenerate: GATTTTAGGAGGCAAAACG. Primer B is degenerate
with the sequence T(G/A/T/C)CC(C/T)TC(A/G)AA(C/T)TG(C/T)TC(G/A/T/C)GC. The reaction products were analyzed by agarose gel electrophoresis. The amplified fragments were electroeluted from the gel and subcloned into the pCR®2.1 plasmid (Invitrogen) to generate plasmid pB900.
The nucleotide sequences of the fragments were determined, and the
correctly amplified sequence was identified by comparing the open
reading frames with the N-terminal amino acid sequences of the two ETF subunits.
Nucleotide sequencing was carried out by the dideoxy chain-termination
method (41) with [
-35S]dATP and
Sequenase® version 2.0 with double-stranded template DNA.
Subclones were generated using restriction sites within the cloned
segment of DNA. These plasmids were sequenced using M13 primers
(Sigma). The DNA sequence was used to design synthetic primers, which
in turn were used to sequence the complete fragment. dITP replaced dGTP
as necessary when secondary structure in the DNA made it difficult to
read the sequence.
Identification and Cloning of ETF Subunit Genes--
The DNA
fragment amplified by PCR was used as a probe in Southern blot analysis
and colony hybridization. The probe was labeled with
digoxygenin-11-dUTP (DIG) in a random-primed DNA-labeling reaction
(Boehringer Mannheim kit, catalogue number 1175033). Purified genomic
DNA was digested with restriction enzymes both singly and in pairs to
generate DNA molecules that could be cloned into pBluescript
SK+ (Stratagene). The restriction digests were separated by
electrophoresis in 0.8% agarose gel and transferred to nylon membrane
(Biodyne® (42). DNA fragments that contained the genes
that encode ETF were identified by direct hybridization of the blots
with the DIG-labeled probe and subsequent detection in the specific
antibody-mediated chemiluminescence reaction. Larger amounts of DNA
were digested with the enzymes that produced fragments of DNA that
hybridized to the probe and were likely to contain the full-length
genes. Size-selected fragments were isolated from agarose gel and
ligated with pBluescript SK+ that had been digested with
the same enzymes. The ligation products were transformed into E. coli TG1, and the colonies that contained recombinant plasmids
were screened by colony hybridization using the DIG-labeled probe.
Plasmid DNA was isolated from transformants that contained DNA that
hybridized to the probe. These plasmid preparations were than screened
by restriction mapping analysis and DNA sequencing. One plasmid with a
2.1-kb insert was found to contain the complete genes for ETF except
for the 5'-end that encodes the N terminus of the
-subunit. A
plasmid with the full-length genes was constructed by ligating this
insert with the 5'-end of pB900 and pBluescript SK+. The
plasmid was termed pBETFfl.1.
Expression of Recombinant Genes--
E. coli was
grown either aerobically or anaerobically at 37 °C in the presence
or absence of IPTG as indicated. Crude lysates of E. coli
were made by resuspending the cell pellets in lysis buffer (50 mM Tris-HCl, 40 mM EDTA, pH 8.0), to which
lysozyme was added (1 mg/ml). After incubation on ice (30 min) and
centrifugation (18,500 × g, 20 min, 4 °C), the
supernatants were analyzed by native PAGE using 10% gels and a
discontinuous buffer system (39). The gels were stained for NADH
dehydrogenase activity using 0.01 mM
p-iodonitrotetrazolium violet and 0.1 mM NADH in
0.1 M potassium phosphate buffer, pH 7.
Computer-based Methods--
DNA sequence analysis was carried
out using the Macmolly package (Softgene, Berlin). The amino acid
sequences were aligned using ClustalW (43). The secondary structure of
polypeptide sequences was determined using Predict Protein
(44-46).
 |
RESULTS |
Cloning and Sequence of ETF Genes--
The relative physical
proximity and arrangement of the genes for the two ETF subunits within
the M. elsdenii genome was not known at the beginning of the
investigation. The N-terminal sequences of the
-subunit (30 residues) and the
-subunit (32 residues) were determined with the
intention of using them to generate primers for PCR. However, a
sequence similarity search of the protein/DNA data bases using the
BLAST search algorithm (47) revealed that the N-terminal sequence of
the
-subunit of M. elsdenii ETF is encoded by a region
downstream from the gene encoding butyryl-CoA dehydrogenase
(bcd) (48). The assumption was then made that the gene for
the
-subunit (etfA) would occur directly downstream from
the
-subunit gene. Consequently, two oligonucleotide primers were
designed and synthesized for use in a PCR amplification of the ETF
genes. Primer A was derived from the sequence of bcd (48), beginning 22 bases upstream from the initiation codon of
etfB. Primer B was degenerate (256-fold), and it
corresponded to the amino acid sequence of the
-subunit from residue
19 to 13. The amplified products were cloned into pCR®2.1
(Invitrogen). Nucleotide sequencing showed that a 900-base pair PCR
product contained base sequence that corresponded to parts of the two
ETF subunits; this was designated pB900.
The sizes of the subunits of ETF predicted that the complete ETF genes
would occur on a 2.1-kb fragment of DNA. A Southern blot was carried
out with M. elsdenii genomic DNA that had been digested with
restriction enzymes that were selected from restriction maps of the
base sequences for bcd (48) and pB900. Size-selected fragments (2.0-2.2 kb) produced from the digestion of genomic DNA with
PstI and HindIII were ligated into pBluescript
SK+ to produce a subgenomic DNA library. The labeled 0.9-kb
PCR product was used to screen the library. Of 2059 colonies screened,
38 were found to contain DNA that hybridized to the probe. One of these
plasmids, with a 2.1-kb PstI-HindIII insert, was
found to contain the complete genes for ETF except for the 5'-end that encodes the N terminus of the
-subunit. A plasmid that contained the
full-length genes was constructed by ligation of the 2.1-kb fragment of
DNA together with the 5'-end of pB900 and pBluescript SK+.
This plasmid was designated pBETFfl.1.
The partial restriction map and sequencing strategy used to establish
the complete nucleotide sequence of the cloned fragment of DNA is shown
in Fig. 1. Each base in the 2102-base
pair fragment was sequenced an average of 2.96 times. The amino acid
sequence of the
-subunit was derived from bases 23-832 (Fig.
2). A putative ribosome-binding site
similar to those found in E. coli is positioned 10 bases
upstream from the initiation codon. No other possible start sites for
translation occur upstream from the ATG codon. The gene is terminated
by a single TAA stop codon. It encodes a polypeptide with 270 amino
acids and a molecular mass of 29,081 Da. The gene etfA that
encodes the
-subunit of ETF is separated from etfB by 20 bases. The amino acid sequence
-ETF was derived from bases
856-1869. This gene is also preceded by a putative ribosome-binding
site (bases 838-845), 10 bases upstream from the ATG initiation codon,
and it is terminated by a single TAA stop codon. It encodes 338 amino
acids, and the polypeptide has the molecular mass 36,101 Da. The two
genes are translated in different reading frames, indicating that
although they might be transcribed as a single polycistronic mRNA
molecule, they are translated as individual polypeptides similar to
other bacterial ETFs. A nucleotide sequence that resembles a
-independent transcription termination site that occurs in E. coli is located downstream from etfA between bases 1887 and 1925 (Fig. 2). Although several stem-loop structures were
identified, only one fulfills the requirements of a
-independent
terminator (49). The free energy of formation calculated for this RNA
hairpin structure using the program Mfold (50) is
15.1 kcal/mol. This
value is similar to values calculated for similar structures in
E. coli (51). The G/C content of etfA and
etfB is 50.15 and 49.45%, respectively. There is a slight bias in favor of G (16.8%) and C (37.4%) at the third position of
codons as reflected in the G/C content of M. elsdenii
genomic DNA (53.6%).

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Fig. 1.
Partial restriction map and sequencing
strategy used to sequence the M. elsdenii genomic DNA
fragment that encodes the ETF subunits. The location of the open
reading frames that encode the - and -subunits are indicated by
and , respectively. The arrows indicate the direction
and extent of the various sequencing reactions. Subclones 1S, 1M, 2M,
and 3B were constructed for sequencing. The positions are indicated for
the restriction sites EcoRI, PstI,
EcoRV, BglII, and HindIII that were
used in the subcloning procedures.
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Fig. 2.
Nucleotide sequences and deduced amino acid
sequences of the etfA and etfB genes of
M. elsdenii. Putative ribosome-binding sites are
underlined. A potential -independent transcription
terminator is indicated by convergent
arrows.
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Amino Acid Sequence Comparisons--
The BLAST search algorithm
(47) was used to search the GenBankTM, Swiss-Prot, and PIR
data bases for amino acid sequences that show similarity to the amino
acid sequences of the subunits of M. elsdenii ETF. The two
subunits were treated as separate polypeptides and used independently
to search the data bases. Earlier analyses identified similarity
between ETF-like sequences from eukaryotes and prokaryotes (14, 52,
53). The present analysis shows that the sequences of the subunits of
M. elsdenii ETF are similar not only to these sequences, but
also to additional sequences that have become available more recently
(Table I). They are most similar to
putative ETFs from the anaerobic bacteria Clostridium acetobutylicum and Clostridium thermosaccharolyticum.
The genetic organization of the etfA and etfB
genes in all three organisms is also similar, suggesting that the
proteins have similar physiological functions. All of these ETF genes
resemble the fixAB genes of N2-fixing bacteria.
The fixAB genes are proposed to function in nitrogen
fixation, but their role has not been established, nor have they been
investigated at the protein level. It is interesting that although
M. elsdenii does not fix nitrogen, its ETF resembles the
fixAB gene products more closely than the well characterized ETFs from other bacteria and mammals.
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Table I
Comparison of the amino acid sequences of ETF and ETF-like proteins
with the subunits of M. elsdenii ETF
The percentages of sequence identities to the subunits of M. elsdenii ETF are shown.
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Confirming previous analyses (14, 53), multiple alignment of the amino
acid sequences of the larger of the two subunits of ETF and FixB
polypeptides shows that there is much sequence similarity between the
C-terminal halves of these proteins but little similarity toward the N
terminus. In contrast, the corresponding alignment for the small
subunits shows that similarities occur throughout the polypeptide.
The FAD-binding site of human ETF involves mainly the C-terminal region
of the
-subunit (6). Eighteen residues occur within hydrogen bonding
distance of the FAD molecule, and of these, 16 are contributed by the
-subunit and 2 by the
-subunit. Fourteen of the residues are
conserved in M. elsdenii ETF (Fig.
3A), making it likely that one
of the two FAD molecules is bound at this site. Two substitutions occur
for residues that in the human enzyme interact with the isoalloxazine
moiety; residue His
286 (alignment position 301) that
bonds to the oxygen of the carbonyl at C(2) of the flavin is replaced
by proline in M. elsdenii ETF (Pro
287), and
residue Tyr
16 (not shown) whose side chain is 3.6 Å from the methyl group at flavin C(8) in the human protein is replaced
by Thr
13 in M. elsdenii ETF. The residue
His
286 is within 2.9 Å of the carbonyl oxygen at C(2)
of the isoalloxazine moiety in the human protein and is postulated to
function in the stabilization of the anionic semiquinone (6). The
substitution of this residue by a proline in the M. elsdenii
enzyme would presumably introduce a bend into the protein backbone and
also remove a positive charge close to isoalloxazine moiety. Removal of
the positive charge may account for the observation that stabilization
of the anionic semiquinone during reductive titration of M. elsdenii ETF is only kinetic, while in other bacterial ETFs and
mammalian ETF both kinetic and thermodynamic stabilization of the
anionic semiquinone occurs.

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Fig. 3.
Amino acid sequence alignments of ETF and
ETF-like sequences around the residues involved in binding the AMP and
FAD in the -subunit (A) and -subunit (B)
of human ETF. The abbreviations used are as in Table I.
Me., M. elsdenii. The sequences were aligned
using ClustalW. A dash indicates a position at which a gap
is inserted to optimize the alignment; an asterisk indicates
a residue that is invariant; a colon indicates a residue
that is highly conserved; a period indicates a residue that
is moderately conserved in the sequences. ¥, , and indicate
residues that are involved in binding the isoalloxazine, ribityl chain,
and AMP moieties of FAD, respectively. ± indicates a residue that
interacts with AMP in the -subunit. The residue numbers in the
alignment sequence are given, with the numbers for M. elsdenii ETF in parentheses (see Fig. 2). The open
reading frames of the fixA genes from E. coli
k12, 1 min and 32 min, are longer than those given above (63); we
suggest that their initiation codons were incorrectly assigned and that
methionine at alignment position 7 is the correct start codon.
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The other two replacements that occur are for residues involved in
binding the AMP part of FAD in the human enzyme, and both are
conservative changes: Lys
301 (alignment position 239) to
Asn
225 and Leu
319 (alignment position
343) to Val
320. As has been identified in other ETFs
(14), this part of the M. elsdenii ETF sequence is similar
to a fingerprint sequence that is diagnostic of a 

fold that
is known to be involved in the binding of the ADP moiety of FAD and
nicotinamide dinucleotides (Fig.
4A) (54). Its consensus
sequence has three highly conserved glycine residues flanked by small
hydrophobic residues, with an acidic residue occurring at
the C terminus of the sequence (55): (V/I/L/A)3G(A/G/S)G(A/G/I/L/S)2G(A/S)(A/G/I/S/V)X12(F/I/L/M/V)(D/E). The first two glycines of the consensus sequence are alanines in
the M. elsdenii sequence. The replacement of the first
glycine by alanine occurs in other ETF and ETF-like sequences examined (6, 14) and also in alcohol dehydrogenase from yeast (56). The
substitution for the second glycine of the consensus sequence has not
been noted before in an FAD-binding protein. An aliphatic residue in
the N-terminal end of the consensus sequence is replaced by tyrosine in
M. elsdenii ETF, as also occurs in other FTFs and in
glutathione reductase of human erythrocytes (57). Furthermore, the
secondary structure of the residues surrounding these amino acid
residues is in the form of a 

fold, which is diagnostic of a
flavin-binding site (Fig. 4A).

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Fig. 4.
Amino acid sequences in the -subunit of
M. elsdenii ETF that are similar to the
dinucleotide-binding consensus sequence for FAD/NAD(P)H-binding
proteins. A and B show the proposed
dinucleotide-binding sites in the -subunit. The amino acid positions
according to the sequence of M. elsdenii ETF are given (see
Fig. 2). The secondary structure was derived using Predict Protein
(46-48). H, E, and L represent
-helix, -sheet, and loop, respectively. Only secondary structures
that are predicted with a reliability >6 (scale 0-9) are shown.
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The sequence similarities with the human enzyme around the FAD-binding
site and the presence of a dinucleotide-binding motif provide strong
evidence that an FAD binds in this part of the M. elsdenii
sequence. However, two additional dinucleotide-binding sites are likely
in M. elsdenii ETF, one for the second molecule of FAD and
one for NADH. A possible site was identified close to the putative
FAD-binding site described above toward the N terminus of the
-subunit (Fig. 4B). In this second sequence, only the
first Gly of the GXGXXG motif is conserved.
However, the sequence is similar to that of the NAD(P)H binding site of mercuric reductases (55). The second Gly is replaced by a serine, and
the third Gly is replaced by an alanine, as also occurs in the NAD(P)H
binding site of glutathione reductases from E. coli and
humans (55). A hydrophobic residue replaces the C-terminal acidic
residue of the consensus sequence. Again, a similar substitution occurs
in the NAD(P)H binding site of mercuric reductases. It is possible that
this site in M. elsdenii ETF binds FAD or NADH. While at
first sight it appears unlikely that two FAD sites could involve two
sets of amino acids so close in the sequence, there is spectroscopic
evidence that the two flavins are not too far apart. Thus, binding of
the second FAD to ETF causes an unexpectedly large increase in
absorbance around 405 nm (Ref. 24; see later), and measurements by
time-resolved polarized fluorescence anisotropy decay suggest that the
centers of the isoalloxazine moieties of the two flavins are 12-23 Å apart (58). The secondary structure around the residues at the second
putative dinucleotide-binding site on the
-subunit is not predicted
to be in the form of a 

fold. If FAD is bound at this site,
the lack of a classic nucleotide-binding fold might result in weaker
binding of the flavin and its consequent loss during enzyme isolation.
It should be noted, however, that the human enzyme does not bind a
second FAD but that it shares much sequence similarity in this region with M. elsdenii ETF.
The M. elsdenii subunit sequences were also analyzed for two
different motifs known to be associated with the binding FAD and/or NAD
in other proteins. One of these is a proline-rich sequence that is
thought to be involved in binding the AMP moiety of FAD in human
ETF-CoQ oxidoreductase and in succinate dehydrogenase from E. coli, yeast, and ox (57). The other is a sequence that is common
in flavoprotein hydroxylases with a putative dual function in binding
FAD/NAD(P)H molecules (59). Sequences that match these motifs do not
occur in M. elsdenii ETF.
The AMP molecule in the human enzyme is buried in the
-subunit. The
eight residues in human ETF that are in hydrogen bonding distance of
the AMP molecule were located using Insight II (60). Three of these
residues are completely conserved in the M. elsdenii enzyme,
and one other residue occurs as a conservative substitution (Fig.
3B). In addition, much similarity between the human and M. elsdenii proteins occurs in residues surrounding these
positions. Although M. elsdenii ETF does not bind AMP (26),
it is possible that it binds either the second FAD at this site or the
substrate NADH. The residues concerned are also moderately conserved in other
-ETFs and in FixA sequences, suggesting that these proteins also may have a dinucleotide-binding site in this region.
It was of interest to determine whether the amino acid sequence
similarities between ETF and the other proteins of Fig. 3 extend to
structural homology. Predictions of secondary structure were made for
the two subunits of ETF from M. elsdenii and were compared
with those predicted by the same method for human ETF as well as those
observed in the crystal structure. Predictions were also made for the
FixAB polypeptides of Azorhizobium caulinodans, and these
are included for comparison (Fig. 5). The
secondary structures determined for the human enzyme by theoretical
analysis of the amino acid sequence with Predict Protein (44-46) are
in good agreement with those determined from the three-dimensional structure (6, 61), thus providing confidence in the theoretical analysis of sequences for which three-dimensional structures are not
available. The computer program used to analyze the amino acid
sequences recognizes
-helices or
-sheets and loop regions, but it
does not recognize 3/10 helices or
-bridges. A bend or a H-bonded
turn observed experimentally appears as a loop in the theoretical
analysis.

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Fig. 5.
Comparison of the predicted secondary
structures in the ETFs from M. elsdenii and human and the
fixAB-derived polypeptides from A. caulinodans.
A shows a comparison of secondary structure of -ETFs from
M. elsdenii (Me) and humans (HsA from the crystal
structure; HsB from the amino acid sequence) and FixB from A. caulinodans (Ac). B shows a comparison of
-ETFs and FixA polypeptides. The secondary structure was derived
using the Predict Protein program (46-48). H, E,
and L represent -helix, -sheet, and loop,
respectively. Only secondary structures that are predicted with a
reliability >6 (scale 0-9) are shown. The secondary structure in the
crystal structure of human ETF (HsA) was derived using the "sstruc"
module of Procheck (61). B, -bridge; E,
-sheet; G, 3/10 helix; H, -helix;
S, bend; T, hydrogen-bonded turn; e,
extension of -sheet; g, extension of 3/10 helix;
h, extension of -helix. An asterisk indicates
a position at which the amino acid is identical. A dash
indicates a position where a gap was inserted to optimize the alignment
of the amino acid sequences.
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The two subunits of human ETF comprise three separate domains: domain
I, the N-terminal part of the
-subunit; domain II, the C-terminal
part of the
-subunit and a small C-terminal part of the
-subunit;
and domain III, composed of most of the
-subunit (6). As noted
above, the sequences of the larger polypeptides (
-ETF/FixB) are
identical at many positions in domain I, and this is reflected in the
very similar secondary structure predicted for this region.
Furthermore, the proteins from the three different sources are all
predicted to have a Rossmann fold (62) at the conserved amino acids
that in human ETF are involved in binding the ADP moiety of FAD (Fig.
5A; alignment positions 294-329). Very little sequence
identity occurs in domain II, indicating that a more rapid sequence
divergence has occurred in this domain. Nevertheless, the secondary
structure predicted for this region is almost identical for the three
proteins, suggesting that the function of domain II is common to all
three proteins.
The secondary structures of the small subunits (domain III) are also
strikingly similar throughout their sequences (Fig. 5B). The
conserved sequences around the AMP-binding site of the human protein
(alignment positions 121-155) have a 

structure that is
similar to a Rossmann fold, a feature that is not revealed by analysis
of the amino acid sequences. As described above, it is possible that in
M. elsdenii ETF this is either the NADH binding site or the
site at which the second FAD binds. Furthermore, a loop (alignment
positions 227-235) has been extended in the M. elsdenii
sequence that may allow greater access to such a dinucleotide-binding site. The loop is also extended in the A. caulinodans
sequence, with the implication that this enzyme may also bind a second
dinucleotide and/or catalyze the oxidation of NADH.
Expression of Recombinant ETF--
An overall aim of the present
investigation was the expression of the recombinant genes for M. elsdenii ETF in E. coli and purification of the
recombinant protein in an amount sufficient for biochemical
characterization. The genes that encode M. elsdenii ETF were
inserted into pBluescript in the appropriate orientation for
transcription to be controlled by the lac promoter
(pBETFfl.1). The native ribosome-binding sites are located upstream
from each subunit gene. However, a promoter region is not present. It
was shown that the plasmid is lost from E. coli during
culture at 37 °C with the inference that the plasmid or its gene
product is toxic to the cells. An enzyme stain that measures diaphorase activity showed that when extracts of E. coli are analyzed
by PAGE, expression of M. elsdenii ETF is not detectable in
cultures grown aerobically with or without IPTG (0.1-1.0
mM). However, when E. coli is grown
anaerobically, the plasmid is retained by the cells, and recombinant
protein is expressed at a low level. The level of expression is
increased by the addition of IPTG to early log phase cultures,
indicating that the genes are under the control of the vector promoter.
Maximal expression of protein was observed with 0.5 mM IPTG
(data not shown).
Purification and Properties of Recombinant ETF--
Recombinant
M. elsdenii ETF was purified from an extract of E. coli TG1 (pBETFfl.1) that had been grown anaerobically (Table II). The final preparation gave only two
bands of protein after SDS-PAGE, indicating that the protein was at
least 95% pure (Fig. 6). The absorbance
ratio, A272:A450, was
6.1, similar to that reported for native ETF (24). The yield of pure
protein was approximately 1 mg/g of E. coli cell paste, and
it is therefore greater than the yield of native ETF from M. elsdenii (0.27 mg/g cell paste). The molecular masses of the
subunits of the recombinant protein were estimated from SDS-PAGE
analysis to be 35.5 and 29.5 kDa for the
- and
-subunit,
respectively (Fig. 6). These values are in close agreement with those
calculated from the predicted amino acid sequence (36.1 and 29.1 kDa).
The electrophoretic mobilities of native and recombinant ETF are
identical (Fig. 6). Hence, the somewhat greater values reported earlier
for the subunit molecular masses of the native form of this ETF are
erroneous (41 and 33 kDa; Ref. 24).
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Table II
Purification of recombinant M. elsdenii ETF
The starting material was 77 g of E. coli cell paste.
The diaphorase assay was used to measure enzyme activity.
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Fig. 6.
SDS-PAGE of recombinant ETF at different
stages of purification. Top, SDS-PAGE gel after
staining with Coomassie Blue. Lane 1, molecular
mass markers; lane 2, crude extract;
lane 3, after Q-Sepharose chromatography;
lane 4, after phenyl-Sepharose chromatography;
lane 5, native ETF; lane 6,
molecular mass markers. The marker proteins were in order of decreasing
size: bovine serum albumin, ovalbumin, glyceraldehyde-3-phosphate
dehydrogenase, carbonic anhydrase, trypsinogen, trypsin inhibitor, and
-lactalbumin. Bottom, determination of subunit mass. ,
molecular mass markers; , ETF subunits (from lanes 4 and
5 above).
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The absorption spectrum of the recombinant ETF has maxima at 273, 373, and 451 nm and minima at 314 and 398 nm. These values agree closely
with those determined for native ETF in the present investigation and
with those reported earlier (24) (
max = 272, 375, 450, and 660 nm;
min = 312 and 398 nm) (Fig.
7). However, some differences occur
between the spectra of the different preparations. The absorbance at
660 nm of the native enzyme is much greater that than of the
recombinant protein. The spectrum of the native enzyme shows pronounced
shoulders at 430 and 510 nm as well as higher absorbance at 400 nm
compared with that of the recombinant enzyme. These features indicate
that the preparation of native enzyme contained hydroxylated flavins
(24). The long wavelength absorbance at 660 nm is characteristic of
6-OH-FAD (green), and the shoulder at 510 nm is characteristic of ETF
that contains 8-OH-FAD (orange) (27). It is clear that the recombinant
protein contains little if any hydroxylated FAD.

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Fig. 7.
Optical spectra of ETF. The proteins
were dissolved in 100 mM potassium phosphate, pH 6, 0.3 mM EDTA. Solid line, isolated native ETF;
dashed line, recombinant ETF as isolated; dotted
line, recombinant ETF after saturation with FAD. The spectra have
been normalized at 450 nm.
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The absorption spectrum of the flavin extracted by heat treatment of
recombinant ETF was found to be identical to that of authentic FAD. The
extinction coefficient of the bound flavin was calculated to be 11,300 M
1 cm
1. The value is lower than
reported for native ETF (24), but the difference may reflect the
differences in the flavin composition of the two preparations. The FAD
content of the recombinant protein was determined using the absorption
coefficient, protein analysis using the Biuret method, a Biuret
coefficient of 0.833 (24), and a value of 65,958 Da calculated for the
molecular mass of ETF from the predicted amino acid sequences of the
two subunits plus one molecule of FAD. The flavin content of the
isolated recombinant ETF was calculated to be 1.06 mol of FAD/mol of
protein. This is lower than the flavin content reported for native
enzyme (approximately 1.4 mol/mol; Ref. 24), a difference that may
reflect the different values for extinction coefficient and molecular
mass determined in the present investigation. The value becomes 1.17 when the extinction coefficient (12,500 M
1
cm
1; Ref. 24) determined earlier is used.
Recombinant ETF binds additional flavin after purification, as also
occurs with native ETF. The binding of additional FAD was monitored by
the difference spectrum between bound and free FAD. This shows maxima
at 405 and 500 nm and minima at 360 and 445 nm (Fig.
8). The flavin-saturated recombinant
protein contains 1.6-1.8 mol of flavin/mol of protein, a value that is
taken to indicate that, as reported for the native enzyme (24),
M. elsdenii ETF contains two FAD-binding sites. The
absorbance at 450 nm of the extra flavin that is bound does not change
relative to that of free FAD, but a large increase occurs around 400 nm
with the result that the trough at 400 nm in the spectrum of isolated
ETF is filled in (Fig. 7). Again, the changes seen with the recombinant protein are similar to those reported earlier for the interaction of
FAD with native ETF (24). The dissociation constant calculated for the
extra FAD bound (0.5-0.6 mol/mol) is approximately 1 µM (Fig. 8). The visible absorption spectrum of the purified recombinant protein does not change when one molar equivalent of FMN or AMP is
added, suggesting that these compounds are not able to replace the
additional FAD.

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Fig. 8.
Difference spectra during titration of
isolated recombinant ETF with FAD. Experiments were carried out
using double sector cuvettes with light paths of 0.45 cm in each
compartment. The front compartment of sample and reference cuvettes
contained the following in 0.75 ml: recombinant ETF as isolated, 22.5 µM in bound flavin; potassium phosphate buffer, pH 6, 0.1 M; EDTA, 0.3 mM. The rear compartments of the
two cuvettes contained the following in 0.75 ml: potassium phosphate
buffer, pH 6, 0.1 M; EDTA, 0.3 mM. A base-line
difference spectrum was recorded before adding increments of 0.84 mM FAD to the front compartment of the sample cuvette and
the rear compartment of the reference cuvette. An equal volume of
buffer was added to the front compartment of the reference cuvette. A
difference spectrum was recorded after each addition of FAD.
Inset, plot of change in absorbance at 405 nm
versus nmol of added FAD.
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The recombinant ETF catalyzes the transfer of electrons from NADH to
BCD and to DCPIP. Specific activities were determined for ETF as it was
isolated and for ETF to which FAD had been added. Two assays were
carried out: a diaphorase assay (coupling the oxidation of NADH to the
reduction of DCPIP) and an ETF assay (coupling NADH oxidation to the
reduction of crotonyl-CoA via BCD) (Table
III). The diaphorase activity of the
isolated recombinant ETF is similar to that determined in the present
work for the native enzyme and also similar to the value reported by
Whitfield and Mayhew (24). The effect of saturating the ETF preparation with FAD seems to vary somewhat with the preparation. Very little effect was observed in the present study and in an earlier report (32);
in contrast, Whitfield and Mayhew (24) reported that the
activity/flavin ratio in this assay decreases when additional FAD
binds.
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Table III
Comparison of the catalytic properties of different preparations of M. elsdenii ETF
Data are given for ETF as it was isolated and for ETF to which FAD had
been added to saturation.
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The activity of recombinant ETF in the ETF assay with BCD is similar to
that of the native enzyme as reported by Whitfield and Mayhew (24). The
activity of the native ETF made in the present work is low by
comparison. These differences probably reflect to some extent the
difficulties inherent to this assay. The activity measured in the ETF
assay depends on the activity of the BCD used, and it is therefore
difficult to compare the activity measurements made in different
laboratories. A 1.5-1.7-fold increase in the activity occurs when
native ETF is saturated with FAD, whereas the change observed with the
recombinant ETF is much smaller. It appears that the additional flavin
that binds has very little effect on the activity of the enzyme in the
ETF assay. The ETF activity of the native preparation used in the
present investigation was low, but the preparation had been stored for several years at
20 °C before assay, and it is possible that a
partial loss in activity may have occurred during storage. Another factor that affects the activities of ETF preparations from M. elsdenii is their content of hydroxylated FAD. Preparations with 6-OH-FAD and 8-OH-FAD have diaphorase activity, but they fail to
catalyze BCD reduction in the ETF assay (25).
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DISCUSSION |
The structural genes that encode the two different subunits of
M. elsdenii ETF have been cloned, sequenced, and expressed in E. coli. Comparison of the deduced amino acid sequences
of the subunits with those of the other well characterized ETFs dispels any doubt that this ETF is a member of the ETF family. However, it has
also been shown that M. elsdenii ETF is more closely related to the putative ETFs of clostridia and to the fixAB gene
products of nitrogen-fixing bacteria. Alignment of the M. elsdenii sequence with that of human ETF provides strong evidence
for a nucleotide-binding site in each of the two subunits. Sequence
homology to the consensus dinucleotide-binding motif and homology to
similar sites in mercuric reductases provide circumstantial evidence
that the third nucleotide-binding site is on the
-subunit. These
tentative conclusions do not support earlier analyses with reactive
flavins and dyes that proposed that all three binding sites would be
found on the
-subunit (30-32). As was pointed out by Roberts
et al. (6) for the human enzyme, the orientation of the two
subunits may mean that binding of modified flavins and dyes to the
-subunit allows their reaction with groups on the
-subunit.
The analysis of the secondary structure in the human, M. elsdenii, and A. caulinodans polypeptides highlights
the structural similarities between these proteins. Although the
overall level of amino acid sequence identity between the proteins is
low, the secondary structural similarities are high throughout the
length of the polypeptides. This suggests that the conformations of the M. elsdenii and A. caulinodans proteins are very
similar to that of the human protein and provides further evidence that
ETF proteins have evolved from a common ancestor.
The genes that encode M. elsdenii ETF are only expressed in
E. coli TG1 when the cells are grown under anaerobic
conditions. The underlying reason for this unexpected finding has not
been investigated. It is possible that M. elsdenii ETF short
circuits electron-transfer pathways in E. coli by coupling
the oxidation of a crucial intermediate such as NADH or a flavoprotein
dehydrogenase to O2. Genes that are similar to the genes
for M. elsdenii ETF have been identified in E. coli (63). It is proposed that they function in electron transfer
to carnitine and that they are only expressed under anaerobic
conditions, lending support to the view that a protein such as M. elsdenii ETF is toxic to aerobic E. coli.
The chemical and catalytic properties of the recombinant protein are
very similar to those reported for native ETF. The differences in the
spectroscopic and enzymic properties of the recombinant and native
proteins are no greater than have been observed between different
preparations of native ETF. They may be due in part to differences in
the flavin composition of the different preparations. The total flavin
in the enzyme isolated varies between 1.06 and 1.4 mol of FAD/mol of
protein, but all preparations bind additional flavin to give
approximately 2 mol/mol of protein. In addition, the content of
6-OH-FAD and 8-OH-FAD varies with each preparation for reasons that are
not yet clear. The AMP that is bound to the
-subunit of human ETF is
thought to have only a structural role in the protein (2). It is
possible that one of the two FAD molecules that binds to M. elsdenii ETF plays a similar structural role.
The function of M. elsdenii ETF is to transfer electrons
from D-lactate dehydrogenase and NADH to butyryl-CoA
dehydrogenase. This ETF may therefore be useful as a model to
investigate inter- and intramolecular electron transfer processes. The
successful cloning of the genes that encode the ETF and their
expression in E. coli will allow a more comprehensive
investigation of the physicochemical and catalytic properties of
M. elsdenii ETF than was possible previously. The native
enzyme has been crystallized (34) with the expectation that it will be
possible to determine the three-dimensional structure of the protein by
x-ray crystallography. The crystal structure should show whether the
possible FAD and NADH binding sites identified in the present study are
correct. In addition, it will be possible to carry out complementary
site-directed mutagenesis to explore the structure and function of the
protein.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF072475.