(Received for publication, December 6, 1994; and in revised form, April 10, 1995)
From the
The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization
and hydroxylation of a precursor, most likely dihydrokalafungin, as the
final steps in its formation. Mutations in the actVB gene
block these last steps, and the mutants secrete kalafungin as a shunt
product. To investigate the role of the actVB gene in these
transformations, we have overexpressed the gene in Escherichia coli and purified and characterized the recombinant protein. ActVB was
shown to catalyze the reduction of FMN by NADH to give NAD and
FMNH
Polyketides comprise a large and diverse family of natural
products, which are built up by the head-to-tail condensation of short
carboxylic acid units, usually acetate or propionate
residues(1) . They are produced by plants, animals, marine
organisms, fungi, and, most prolifically, by actinomycete bacteria.
Their biosynthesis may be divided into two stages: first, the carbon
skeleton is assembled by the requisite polyketide synthase; second, a
variety of ``tailoring'' reactions modify the polyketide
structure by, for example, hydroxylation, glycosylation, or
methylation.
Much interest has focused on the assembly of the
polyketide backbone, which is analogous to the biosynthesis of fatty
acids(1) . The cloning and sequencing of the polyketide
synthase genes for several polyketides has confirmed the analogy with
fatty acid biosynthesis and has been instrumental in advancing our
understanding of the principles of polyketide synthase
programming(2, 3, 4, 5, 6) .
In contrast, the enzymes that catalyze the tailoring reactions are
generally less well understood, even though these later elaborations of
the polyketide structure are usually crucial to the compound's
biological activity. The biosynthesis of actinorhodin involves, as a
late step, an unusual dimerization in which two benzoquinone units are
symmetrically joined by a phenolic oxidative coupling (7) (Fig. 1). Structural features arising from phenolic
couplings are found in a very wide range of natural products, including
the oligopeptide antibiotic vancomycin and opiate
alkaloids(8) , but the enzymic chemistry responsible for them
remains largely unexplored. The cloning and sequencing of the relevant
part of the actinorhodin biosynthetic cluster (9) presented an
opportunity to study an example of this novel enzyme activity using
molecular biology.
Figure 1:
Oxidative
biosynthesis of actinorhodin from dihydrokalafungin. Mutants blocked in actVB secrete kalafungin as a shunt product. The order of the
final (C-8) hydroxylation and dimerization at C-10 is not
established.
To reconstruct the 5`-end of the actVB
gene, two oligonucleotides, TATGGCTGCTGACCAGGGTATGCTGCGTGACGCTATGGCT
and GAGCCATAGCGTCACGCAGCATACCCTGGTCAGCAGCCA, corresponding to sense and
antisense strands, respectively, were synthesized. These
oligonucleotides both optimized codon usage for highly expressed E.
coli genes (16) and allowed cloning into the NdeI
site of the pT7-7 vector. The oligonucleotides were
phosphorylated, annealed together, and ligated into pACTVB-NT
previously digested with NdeI, EcoRI, and SmaI (EcoRI digestion served to prevent the
polylinker fragment religating into pACTVB-NT). Transformants were
selected by plating on 2TY agar containing 100 µg/ml ampicillin. To
confirm that the actVB gene had been correctly reconstructed,
plasmids were isolated from the recombinant bacteria, and the
nucleotide sequence of the first 100 base pairs of the 5`-region was
determined. The plasmid containing the actVB gene under the
control of the T7 promoter was designated pACTVB.
The dialysis
residue was cleared by centrifugation (25,000
ActVB protein could be further purified on a small
scale by dye ligand chromatography. Nine units of ActVB in 0.5 ml of 20
mM potassium phosphate buffer, pH 6.4, were applied to a small
(2 ml) column of Amicon Matrix Blue A equilibrated in the same buffer.
The protein was allowed to bind to the column for 30 min, and then
proteins that remained unbound were removed by washing with 10 ml of
phosphate buffer. ActVB was eluted with 1.5 M potassium
chloride in 20 mM potassium phosphate, pH 6.4.
Anaerobic assays were performed in gas-tight cuvettes under argon.
Solutions were de-oxygenated by bubbling through with argon gas that
had been scrubbed free of oxygen.
Figure 2:
Subcloning strategy for the construction
of pACTVB. Inset, the protein sequence, the Streptomyces
coelicolor 5`-DNA sequence, and the replacement 5`-oligonucleotide
sequence (coding strand) for the N-terminal part of ActVB. Nucleotides underlined represent those changed during oligonucleotide
synthesis for optimal codon usage in E.
coli.
After purification by dye column chromatography, ActVB
was very nearly pure as judged by SDS-PAGE (Fig. 3). The two
major protein bands seen on the gel corresponded to different forms of
ActVB (as discussed below), and minor contaminating bands of M
Figure 3:
Purification of ActVB. SDS-PAGE of samples
taken after each step of the purification (gels stained with Coomassie
Brilliant Blue) is shown. Lane1, soluble cell
fraction; lane2, ammonium sulfate fractionation; lane3, pooled fractions after gel filtration
chromatography; lane4, pooled fractions after ion
exchange chromatography; lane5, protein after dye
ligand chromatography. The positions and molecular weights of marker
proteins are indicated at the sides of the
gel.
During the expression of
ActVB, a protein of lower molecular mass (
The site of proteolysis was
investigated by electrospray mass spectrometry. The mass spectrum of a
mixture of the two ActVB forms showed two major series of peaks. One of
these was due to a species of M
Figure 4:
Reduction of FMN catalyzed by ActVB. i, UV-visible spectrum before addition of enzyme; ii,
after complete reduction by NADH.
Figure 5:
Hanes plot of kinetic data for ActVB.
Enzyme activity was measured at fixed concentrations of ActVB and
varying concentrations of NADH and FMN. The concentrations of FMN used
were 40 µM (
The cloning and sequencing of the actinorhodin biosynthetic
gene cluster has afforded the opportunity to study in detail the
biochemistry of polyketide assembly. ActVB is the first enzyme from
this pathway to be overexpressed in E. coli and physically and
kinetically characterized. The actVB gene was originally the
focus of our investigation because mutations mapping to this gene
abolished two chemically interesting steps, the final hydroxylation and
dimerization, the enzymology of which is little explored.
Initially,
we thought that ActVB might catalyze either the hydroxylation or
dimerization of kalafungin or dihydrokalafungin directly. However,
extensive trial assays using a variety of different buffers, pH, metal
ions, cofactors such as flavin, and both oxidized and reduced
nicotinamide coenzymes failed to produce any evidence that ActVB could
catalyze the hydroxylation or dimerization of either potential
substrate. The initial suggestion that ActVB might be an oxidoreductase
came from sequence similarity observed between ActVB and a component of
pristinamycin IIB hydroxylase
We
subsequently confirmed that ActVB is a flavin:NADH oxidoreductase; the
preferred substrate is FMN, but FAD and riboflavin are effective
substrates at high concentrations. Since it was not possible to purify
ActVB to homogeneity, the enzyme activity might possibly result from a
contaminating protein. However, we feel this is unlikely because of the
sequence similarity to SnaC and the fact that ActVB binds FMN
stoichiometrically. Unusually, FMN behaves as a true substrate in this
reaction rather than as a tightly bound cofactor. This is confirmed by
the absence of any absorption bands attributable to flavin in the
UV-visible spectrum of the purified protein, the quantitative reduction
of substrate levels of FMN under anaerobic conditions in the presence
of excess NADH, and steady state kinetic analysis, which clearly
indicates the formation of a ternary complex of FMN and NADH with
ActVB, implying direct reduction of FMN by NADH.
The only well
studied example in which free FMNH
The order of the hydroxylation and
dimerization steps that transform dihydrokalafungin to actinorhodin is
unknown. On simple chemical grounds, dimerization was favored as the
first step since the phenolic hydroxyl at C-11 would direct coupling to
the ortho (C-10) position. Accumulation of kalafungin by the actVB mutants was therefore taken as evidence that they were
blocked in dimerization itself(9, 10) . However,
hydroxylation could precede dimerization if the dimerase could
distinguish between the two para-substituted hydroxyl groups
(at C-8 and C-11) and so direct the regiochemistry of coupling to C-10
rather than C-9. Evidence that one or more genes essential for C-8
hydroxylation are located in the actVA region of the act cluster (far from the actVB gene) came from the finding
that this DNA segment could cause Streptomyces sp. AM 7161,
the producer of medermycin, which lacks the C-8 hydroxyl, to make
mederrhodin A, which is hydroxylated at C-8(25) . Therefore, if
ActVB is indeed involved in this hydroxylation step, as suggested by
its similarity to HpaC and SnaC, Streptomyces sp AM 7161 must
carry an equivalent gene.
There are many examples in which reduced
flavin is used to activate dioxygen toward hydroxylation chemistry,
which occurs through the intermediacy of a flavin 4a
hydroperoxide(26) . The chemistry of the dimerization is more
speculative. Dihydrogeodin oxidase, one of the few enzymes catalyzing
oxidative couplings that have been characterized, requires copper and
uses molecular oxygen as the oxidant(27) . However,
flavin-mediated redox chemistry is sufficiently versatile that reduced
FMN might well also play a role in the oxidative dimerization leading
to actinorhodin. We are currently investigating genes within the actVA region of the actinorhodin biosynthetic cluster to
identify the hydroxylase and dimerase enzymes and to clarify the role
of FMNH
We thank Francisco Malpartida (Centro Nacional de
Biotecnologia, Madrid) (who provided pIJ2347), Maureen Bibb, and Peter
Revill (John Innes Centre) for helpful discussions. We also thank Paul
Skelton (Dept. of Chemistry, University of Cambridge) for protein
analysis by electrospray mass spectrometry and Mike Wheldon of the
Protein and Nucleic Acid Facility (Dept. of Biochemistry, University of
Cambridge) for the synthesis of oligonucleotides and protein sequence
determination.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
, which, unusually, is released into solution. The
protein contains no chromogenic cofactors and exhibits no requirements
for added metal ions. The reaction obeys simple kinetics and proceeds
through the formation of a ternary complex; K
values for FMN and NADH are 1.5 and 7.3 µM,
respectively, and k
is about 5
s
. FAD and riboflavin are also substrates for the
enzyme, although they have much higher K
values. The subunit structure of the enzyme was investigated
by analytical ultracentrifugation, which showed the protein to exist in
rapid equilibrium between monomer and dimer forms. The possible role of
this oxidoreductase in the oxidative chemistry of actinorhodin
biosynthesis is discussed.
One class of actinorhodin pathway mutants, actVB, is believed to lack the ability to perform the
oxidative coupling step and secrete kalafungin as a shunt product (Fig. 1)(10) . Kalafungin can be converted to
actinorhodin(10) , which suggests that either dihydrokalafungin
or 8-hydroxy-dihydrokalafungin is likely to be the substrate for
dimerization. The two available actVB mutations have been
mapped to a single open reading frame encoding a protein of 177 amino
acid residues (M 18,400) (9) . Here, we
describe the expression of the actVB gene in Escherichia
coli and the purification and characterization of the recombinant
protein, the first enzyme from the actinorhodin biosynthetic pathway to
be characterized biochemically. Initially, we had thought that ActVB
might catalyze the dimerization of either kalafungin or
dihydrokalafungin directly; however, sequence similarity to a component
of pristinamycin IIB hydroxylase
(
)encoded by snaC (11) and successful complementation of an actVB mutant by snaC
(
)suggested that the protein might function as a
flavin:NADH oxidoreductase. The ActVB protein does indeed catalyze
reduction of FMN by NADH and is therefore probably an essential
auxillary enzyme supplying reduced FMN to another enzyme directly
involved in oxidative chemistry.
Materials
DNA restriction and modifying
enzymes were from Promega.
Isopropyl-1-thio--D-galactopyranoside was from Melford
Laboratories (Ipswich, United Kingdom). Q-Sepharose fast flow anion
exchange medium and Sephacryl S-300-HR gel filtration medium were
purchased from Pharmacia Biotech Inc. Matrex gel Blue A (Cibacron Blue
3GA) was from Amicon. NADH, FMN, FAD, and riboflavin were from
Boehringer Mannheim. pT7-7(12) ,
(
)E. coli TG1 carrying the recO
mutation(13) , and E. coli BL21(DE3) (14) were
the kind gift of Dr. P. Caffrey (Dept. of Biochemistry, Cambridge
University). Kalafungin, nanaomycin, dihydrokalafungin, and
actinorhodin were the kind gift of Prof. S. Omura (Kitasato Institute,
Tokyo).
Construction of Expression Vector for
ActVB
The actVB gene had been subcloned as a 3-kb BglII fragment (encompassing restriction sites 19-21 as
defined in (9) ) in pBR329 to give pIJ2347.(
)pIJ2347 was digested with SmaI and SalI and a 1.1-kilobase fragment encompassing all of the actVB gene, except the first 39 nucleotides isolated and
ligated into SmaI- and SalI-digested and
dephosphorylated pT7-7. The ligation mixture was used to
transform E. coli TG1 recO 1504::Tn5 by standard
methods(15) , and recombinant colonies were selected by plating
on 2TY agar containing 100 µg/ml ampicillin. Recombinant plasmids
were analyzed by restriction mapping; the pT7-7 derivative
containing the actVB gene minus the first 39 nucleotides was
called pACTVB-NT.
Overexpression of Recombinant ActVB
Protein
E. coli BL21(DE3) was transformed with
pACTVB(15) . A 10-ml sample of an overnight culture of a
transformant was used to inoculate each of six 1-liter flasks
containing 500 ml of 2TY media supplemented with 50 mM potassium phosphate, pH 6.9, 10% glycerol, 0.4% glucose, and 100
µg/ml ampicillin. Cultures were grown at 37 °C until an A of 3.5 was reached. These cells were
harvested by centrifugation and resuspended into six 2-liter flasks
containing 1 liter of the same medium but without glucose. Expression
of ActVB was induced by adding 238 mg/liter
isopropyl-1-thio-
-D-galactopyranoside, and the cells were
grown for a further 2.5-3 h (A
4.0). Cells were harvested by centrifugation, washed with 50
mM Tris-HCl, pH 7.4, and stored at -20 °C.
Purification of Cloned ActVB Protein
All
steps were performed on ice or at 4 °C. In a typical purification,
26-g cells (damp weight) were thawed on ice and resuspended in 50 ml of
buffer A (50 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 10% glycerol). Cells were ruptured by passing them twice
through a French pressure cell operating at 15,000 p.s.i. internal
pressure, and cell debris was removed by centrifugation (25,000 g for 15 min). Nucleic acids were precipitated by the dropwise
addition of 20% streptomycin sulfate solution to a final concentration
of 4% and were removed by centrifugation (25,000
g for
15 min). The supernatant was then brought to 20% saturation by the slow
addition of 107 g/liter solid ammonium sulfate. Proteins precipitated
by this step were removed by centrifugation (25,000
g for 15 min), and the supernatant was brought to 60% saturation by
the addition of a further 244 g/liter solid ammonium sulfate. The
precipitated protein was recovered by centrifugation, and the pellet
was resuspended in a minimal volume of buffer A. The protein was
dialyzed overnight against 1 liter of the same buffer.
g for 15
min) and loaded onto a 2.5
100-cm Sephacryl S-300-HR gel
filtration column equilibrated in buffer A. Proteins were eluted at a
flow rate of 28 ml/h, and 3.5-ml fractions were collected. Fractions
were analyzed for the presence of ActVB by SDS-PAGE
(
)and assayed for FMN:NADH oxidoreductase activity as
described below. Pooled peak fractions were applied to a 2.5
15-cm Q-Sepharose fast flow anion exchange column equilibrated in
buffer B (40 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1
mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 10% glycerol) at a flow rate of 60 ml/h. The column was
washed with 300 ml of buffer B, and protein was eluted with a 600-ml
linear gradient of KCl (0-0.3 M) in buffer B. Fractions
(3.7 ml) were analyzed by SDS-PAGE and assayed for FMN:NADH
oxidoreductase activity. Fractions containing flavin reductase activity
were pooled and concentrated by ultrafiltration in a stirred cell
fitted with an Amicon PM10 membrane (exclusion limit, 10 kDa). The
ActVB protein,
80% pure, was stored at -20 °C in the
presence of 20% glycerol. Protein concentration was approximately
8-10 mg/ml.
Protein Analysis
SDS-PAGE was carried out
using the buffer system of Laemmli (17) in a 20% polyacrylamide
gel, and protein bands were visualized by staining with Coomassie
Brilliant Blue R250. The Pharmacia Phast gel system was used with an
IEF5-8 gel to determine the isoelectric point of the protein.
Protein concentrations were determined by the method of
Bradford(18) ; bovine serum albumin was used to construct
standard curves. Recombinant protein identity was confirmed by
N-terminal sequencing of polyvinylidene difluoride-blotted protein on
an Applied Biosystems 477A protein sequencer. Exact protein molecular
weights were determined on a Fission VG Bio Q Electrospray mass
spectrometer. 200 pmol of protein in 20 µl of 50% acetonitrile,
0.1% trifluoroacetic acid were used per injection.
Enzyme Assay
Flavin:NADH oxidoreductase
activity was assayed by monitoring the decrease in absorption of NADH
at 340 nm ( = 6.22
10
M
cm
) in 50 mM Tris-HCl, pH 7.4, at 25 °C. Steady state kinetic measurements
were performed with a 1-cm light path in a final volume of 1 ml. For
routine determination of enzyme activity, 100 µM NADH and
40 µM FMN were present, and assays were initiated by the
addition of enzyme. To maintain enzyme activity, enzyme dilutions were
made into 10% glycerol to give a working stock solution. Solutions
containing FMN were kept in the dark to avoid photoreduction.
Analytical
Ultracentrifugation
Sedimentation velocity measurements
were performed on a MSE Centriscan 75 analytical ultracentrifuge, and
sedimentation equilibrium measurements were performed on a Beckman XLA
machine, using methods previously described (19) . Measurements
were made at 20 °C, and sedimentation was monitored at 280 nm using
scanning absorption optics. Protein samples were made up at a
concentration of 0.5-0.7 mg/ml in TES buffer, pH 7.5.
Expression of ActVB
ActVB was
overexpressed in E. coli by placing the gene under the control
of strong bacteriophage T7 10 promoter(13) . The
subcloning strategy involved reconstructing the 5`-portion of the gene
using synthetic oligonucleotides (Fig. 2), and this allowed the
first 13 codons to be changed to those found in highly expressed E.
coli genes(16) . This has been shown to improve the
expression levels of some Streptomyces proteins, which are
otherwise poorly expressed in E. coli due to the high GC bias
of Streptomyces DNA(20) . Optimal levels of ActVB
expression were achieved by first growing the cells until late
exponential phase (A
3.5-4.0) in
the presence of 0.4% glucose, which represses the basal level of T7 RNA
polymerase expression. Expression was induced by removing them from the
glucose-containing medium by centrifugation and resuspending in fresh
medium containing isopropyl-1-thio-
-D-galactopyranoside.
The cells were grown for a further 2.5-3 h, by which time they
were nearing stationary phase, and expression of ActVB was maximal. The
inclusion of 10% glycerol and 50 mM phosphate, pH 6.9, in the
medium increased the yield of ActVB and may have increased the
proportion of soluble recombinant protein. Typically, 26 g of cells
were produced from 6 liters of medium, and in general the overexpressed
protein comprised 5-10% of total cell protein as judged by
SDS-PAGE.
Purification of ActVB
ActVB was produced
both in soluble form and as inclusion bodies in approximately equal
amounts. The soluble protein, rather than the inclusion bodies, was
purified since it was not known whether the protein could be refolded.
To effect the purification, it was necessary to include 10% glycerol in
all the buffers since aggregation of ActVB proved a considerable
problem, and glycerol appeared to help the protein remain soluble. A
purification based on size exclusion chromatography on Sephacryl
S-300-HR and anion exchange chromatography on Q-Sepharose fast flow
medium resulted in ActVB protein that was more than 80% pure. Removal
of the remaining proteins proved more difficult. Various
chromatographic techniques, including hydrophobic interaction
chromatography on phenyl-Sepharose, fast protein liquid chromatography
using a Pharmacia Mono Q column and affinity chromatography on
FMN-agarose, failed to provide significant further purification.
However, nearly pure ActVB protein could be produced on a small scale
by binding it to a Cibacron Blue 3GA dye column. Although this step
removed nearly all the remaining contaminating proteins, recovery was
only 33% on this step, and the purified ActVB actually had a slightly
lower specific activity. Attempts to scale up this purification step
proved unsuccessful as very substantial losses of protein were
encountered.
30,000 and 35,000 were also still present.
The purified protein was stable when stored at -20 °C in the
presence of 20% glycerol. The yields and specific activities for each
step are shown in Table 1.
ActVB was found to precipitate
irreversibly from solution when the pH was reduced below 6.5, which
indicated a relatively high isoelectric point and restricted the choice
of purification methods. The isoelectric point, calculated from the
protein sequence, is 6.61, and the experimentally determined value by
isoelectric focusing is 6.45. The low yields encountered in the dye
column chromatography step were probably caused by the need to conduct
this step at a pH close to the isoelectric point; at higher pH levels,
ActVB was not bound by the column. Presumably, the losses encountered
when trying to scale up the purification were due to the protein
precipitating irreversibly on the column.
16,000 Da) was also seen (Fig. 3). The smaller protein copurified with ActVB and had the
same N-terminal sequence, indicating that it was derived from ActVB by
proteolysis near the C terminus. The proportion of proteolyzed protein
in the preparation could be reduced by decreasing the length of time
that cells were grown after induction and by the use of proteolytic
inhibitors throughout the purification.
18265 ± 2,
which corresponds to ActVB from which the N-terminal methionine residue
has been cleaved (calculated M
= 18,262).
The other species has an M
of 16,645 ± 2,
which would correspond to ActVB lacking the N-terminal methionine and
cleaved after Cys-158 (calculated M
=
16,642). Since it was not possible to separate the truncated protein
from the full-length form, it is not clear whether it is active or not.
Physical Characterization and Quaternary Structure of
ActVB
Initial estimations of molecular weight made by gel
filtration on a calibrated Pharmacia Superose-12 fast protein liquid
chromatography column at pH 7.5 yielded a M of
32,000 for ActVB, suggesting it to be dimeric. The subunit structure
was investigated in more detail by analytical ultracentrifugation. The
weight-averaged molecular weight over the whole solute distribution, M°
, was determined at several different pH
values between 5.0 and 8.5. M°
decreased
progressively, as the pH was raised, from 31,000 ± 2000 at pH
5.0 to 25,000 ± 1000 at pH 8.5. At pH 7.5, under the conditions
in which gel filtration was performed, M°
for ActVB was 28,000 ± 2000. Only a single symmetrical
boundary was seen during sedimentation velocity, indicating that the
protein exists in a rapid equilibrium between monomer and dimer forms,
with higher pH promoting dissociation. The point weight average
molecular weight extrapolated to zero concentration gave a value of
18,000 ± 5000, which corresponds to the M
of the monomeric protein, as expected for a system that is in
true equilibrium between monomer and dimer. The sedimentation
coefficient, s
, at pH 7.5, was 2.93 ±
0.08 S, a value typical for globular proteins of this size. At lower pH
values, higher molecular weight species were detected at the cell base,
again demonstrating the propensity of the protein to aggregate when
near its pI.
Substrate Specificity and Kinetics
ActVB
prepared from E. coli was colorless, and the UV-visible
spectrum showed no evidence for any chromogenic cofactors.
Oxidoreductase activity depended on both NADH and flavin being added in
the assay; no requirement for any other cofactors was apparent. The
most effective substrates were NADH and FMN, but FAD and riboflavin
could be turned over by the enzyme, although the K values were much higher. Under aerobic conditions, the
oxidation of NADH could be effected with catalytic amounts of FMN,
suggesting that FMNH
was being recycled by reaction with
oxygen to form H
O
and FMN. This was confirmed
when the assay was performed anaerobically. NADH now reduced FMN
stoichiometrically, and the absorption band at 450 nm corresponding to
FMN was completely bleached when excess NADH was added (Fig. 4).
When oxygen was admitted to the assay, further oxidation of NADH took
place, and the absorption at 450 nm due to FMN returned.
The steady
state kinetic properties of the enzyme were investigated; velocities
were measured at 25 °C in 50 mM Tris-HCl buffer, pH 7.4.
The K for NADH was determined at various
concentrations of FMN, and the data were fitted by computer. This
yielded values for the true K
for NADH of
7.3 ± 0.6 µM and K
for FMN of 1.5 ± 0.1 µM. The Hanes plot of
the data (Fig. 5) clearly demonstrates an intersecting pattern
characteristic of the formation of a ternary complex between ActVB,
NADH, and FMN. The specific activity of ActVB was calculated as 8.2
units/mg, which, assuming that both forms of ActVB are active, gives a
value for k
of 5 s
. The
apparent K
values for FAD and riboflavin,
determined at 100 µM NADH, were 11.5 ± 0.6 and 13.5
± 0.6 µM, respectively.
), 20 µM (
), 10
µM (
), 5 µM (
), 2 µM (
), and 1 µM (
).
The stoichiometry of FMN
binding to ActVB was investigated using equilibrium
ultrafiltration(21) . A 10 µM solution of
ActVB protein was incubated in the presence of 50 µM FMN (i.e. a saturating concentration of FMN) for 5 min, and the
solution was subjected to ultrafiltration using a membrane with a
10,000-Da cut off. The concentrations of FMN in the filtrate and
retentate were then determined by the absorbance at 450 nm. The FMN
concentration in the retentate was 58 µM, while that in
the filtrate was 43 µM; therefore, the concentration of
bound FMN was 15 µM. The protein concentration in the
retentate was determined as 17 µM, and hence 88% of the
protein bound FMN. Given that the ActVB preparation was approximately
85% pure, we interpret this as indicating that 1 mol of FMN is bound
per mol of ActVB.
encoded by snaC (11) and, subsequently, successful complementation of an actVB mutant by snaC.
Recently, the
cloning of a FAD:NADH-dependent 4-hydroxyphenylacetate 3-hydroxylase
from E. coli has been reported(22) . The enzyme
comprises two separable protein components, the smaller of which, HpaC,
also has sequence similarity with ActVB. This suggests that HpaC may
similarly function as flavin:NADH oxidoreductase. These enzymes may
comprise a new class of reduced flavin-dependent oxidases.
serves as a cosubstrate
is the oxidation (by molecular oxygen) of aliphatic aldehydes by
bacterial luciferase in luminescent bacteria. FMNH
for this
reaction is provided by FMN:NADH oxidoreductases, of which examples
from Vibrio fischeri and Vibrio harveyi have recently
been overexpressed in E. coli and
characterized(23, 24) . ActVB shows no homology to
these enzymes and differs in structure and kinetic properties. The Vibrio oxidoreductases are monomers of approximately 25,000
Da, while ActVB is dimeric and substantially smaller, with a subunit
mass of 18,400 Da. The Vibrio enzymes contain tightly bound
FMN, distinct from FMN undergoing reduction, and exhibit ping-pong
kinetics, implying that an intermediate reduced flavoprotein is formed.
In contrast, the reduction of FMN by ActVB, as discussed above, appears
to proceed via a ternary complex.
generated by ActVB, the timing of hydroxylation and
dimerization, and the chemistry associated with these transformations.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.