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Journal of Bacteriology, February 2000, p. 627-636, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Functional Analysis of the Small Component of the
4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia
coli W: a Prototype of a New Flavin:NAD(P)H
Reductase Subfamily
Beatriz
Galán,
Eduardo
Díaz,
María A.
Prieto, and
José L.
García*
Department of Molecular Microbiology, Centro
de Investigaciones Biológicas, Consejo Superior de
Investigaciones Científicas, Madrid, Spain
Received 7 September 1999/Accepted 4 November 1999
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ABSTRACT |
Escherichia coli W uses the aromatic compound
4-hydroxyphenylacetate (4-HPA) as a sole source of carbon and energy
for growth. The monooxygenase which converts 4-HPA into
3,4-dihydroxyphenylacetate, the first intermediate of the pathway,
consists of two components, HpaB (58.7 kDa) and HpaC (18.6 kDa),
encoded by the hpaB and hpaC genes,
respectively, that form a single transcription unit. Overproduction of
the small HpaC component in E. coli K-12 cells has
facilitated the purification of the protein, which was revealed to be a
homodimer that catalyzes the reduction of free flavins by NADH in
preference to NADPH. Subsequently, the reduced flavins diffuse to the
large HpaB component or to other electron acceptors such as cytochrome c and ferric ion. Amino acid sequence comparisons revealed
that the HpaC reductase could be considered the prototype of a new subfamily of flavin:NAD(P)H reductases. The construction of a fusion
protein between the large HpaB oxygenase component and the
choline-binding domain of the major autolysin of Streptococcus pneumoniae allowed us to develop a rapid method to efficiently purify this highly unstable enzyme as a chimeric CH-HpaB protein, which
exhibited a 4-HPA hydroxylating activity only when it was supplemented
with the HpaC reductase. These results suggest the 4-HPA
3-monooxygenase of E. coli W as a representative member of
a novel two-component flavin-diffusible monooxygenase (TC-FDM) family.
Relevant features on the evolution and structure-function relationships
of these TC-FDM proteins are discussed.
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INTRODUCTION |
Oxygenases are the enzymes that
catalyze the initial reactions of aerobic catabolic pathways for
aromatic compounds by incorporating either two atoms of molecular
oxygen (dioxygenases) or a single oxygen atom (monooxygenases)
(14, 15). For the monooxygenases that require the NAD(P)H
cofactor, the reaction is separated into two steps, i.e., the oxidation
of NAD(P)H to generate two reducing equivalents and the hydroxylation
of substrates. Most of the monooxygenases catalyzing the hydroxylation
of the aromatic ring are flavoprotein enzymes that carry out the two
reactions on a single polypeptide chain (14, 15). However,
multicomponent monoxygenases where NAD(P)H oxidation and the
hydroxylation reaction are catalyzed by separate polypeptides linked by
an electron transport chain have been also described (14,
15). The most complex monooxygenases described so far are the
six-component proteins for the hydroxylation of aromatic compounds,
such as phenol, benzene, and toluene (5, 14, 15).
Different two-component monooxygenases that hydroxylate aromatic
compounds have been reported and they can be classified in two main
categories according to the nature of the oxygenase component, that is,
as heme and nonheme enzymes. Within the heme monooxygenase group, a
flavoprotein constitutes the electron transfer component, and
cytochrome c or cytochrome P450 is usually found as the
oxygenase component (14, 15). The nonheme two-component
monooxygenases can use either pteridines or flavins as cofactors
(15). In turn, the flavin-dependent nonheme two-component
monooxygenases can be grouped in two major families according to their
electron transfer component. One family comprises those enzymes whose
electron transfer component involves a ferredoxin-NAD(P) domain,
e.g., the diiron XylMA (toluene-xylene monooxygenase) or the
mononuclear iron TsaMB (p-toluenesulfonate monooxygenase)
(19). The other family, referred to hereafter as the
two-component nonheme flavin-diffusible monooxygenase (TC-FDM) family,
comprises several enzymes of uncertain classification reported in the
literature whose reductase component uses NAD(P)H to catalyze the
reduction of a flavin that diffuses to the oxygenase component for
oxidation of the substrate by molecular oxygen.
The 4-hydroxyphenylacetate (4-HPA) 3-monooxygenase from
Escherichia coli W is a two-component enzyme encoded by the
hpaB and hpaC genes and catalyzes the initial
reaction in the degradation of 4-HPA, i.e., the introduction of a
second hydroxyl group into the benzene nucleus at a position
ortho to the existing hydroxyl group, giving rise to
3,4-dihydroxyphenylacetate (3,4-DHPA) (32, 33). This
monooxygenase shows a broad substrate range, hydroxylating phenol
derivatives (32, 33). While the HpaB protein (58.7 kDa) of
4-HPA 3-monooxygenase was shown to be the oxygenase component, HpaC
(18.6 kDa) was assumed to be a coupling protein that enhanced the
activity of HpaB and could prevent the wasteful oxidation of NADH in
the absence of substrate (32). In this regard, a coupling
protein enhancing the activity of an aromatic monooxygenase had been
also described for the 4-HPA hydroxylase from Pseudomonas putida (2, 3). However, in the past four years several
TC-FDM enzymes whose amino acid sequences have revealed significant
similarities with those of the HpaB and HpaC proteins have been
reported in different bacteria, suggesting that HpaB and HpaC could be
considered as the representative oxygenase and reductase components,
respectively, of this new TC-FDM family (13, 16, 21, 36,
41).
In this work, we provide the experimental demonstration that HpaC is
the flavin:NADH oxidoreductase component of the 4-HPA 3-monooxygenase
from E. coli W. Thus, this enzyme becomes the first
sequenced protein in the aromatic TC-FDM family. A comparative analysis
of different members of the rapidly growing TC-FDM family reveals
interesting features on the evolution and the structure-function relationships of these proteins.
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MATERIALS AND METHODS |
Materials.
Restriction endonucleases and phenyl-Sepharose
CL4B, Sephadex G-100, and Superose 12 HR columns were from Pharmacia
Fine Chemicals. Flavin adenine dinucleotide (FAD), flavin
mononucleotide (FMN), NADH, NADPH, 4-HPA, 3,4-DHPA, riboflavin,
cytochrome c, ferrozine, glucose dehydrogenase, Blue
Sepharose and DEAE-cellulose columns, and marker proteins for gel
filtration were purchased from Sigma. Hydroxyapatite-HTP columns and
marker proteins for sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) analysis were purchased from Bio-Rad.
Catalase was purchased from Boehringer Mannheim. Culture media were
obtained from Difco. All other chemicals were of the highest grade
available and were purchased from Sigma or Merck.
Strains, plasmids, media, and growth conditions.
The
E. coli K-12 strains used were DH1 (34) and TG1
(Amersham Pharmacia). Bacteria were grown in Luria-Bertani medium
(34) at 37°C with shaking. The plasmids (and relevant
genotype) used were pUC19 (34), pAJ27 (hpaB)
(32), pAJ28 (hpaC) (32), pAJ22 (hpaB hpaC) (32), and pCE17
(c-lytA) (35). It is worth noting that the
hpaC and hpaB genes were expressed in E. coli K-12 strains which lack the 4-HPA degradative cluster in
their genomes (33).
DNA manipulations and transformation.
Plasmid preparation
and isolation of DNA fragments were carried out by standard procedures
(34). Restriction endonucleases were used according to the
manufacturer's instructions. Transformations of E. coli
cells were carried out by the RbCl method (34). Nucleotide sequences were determined directly from plasmids by using an ABI-377 automated DNA sequencer (Applied Biosystems, Inc., Foster City, Calif.).
Computer analyses.
Protein sequence similarity searches were
made by using the BLASTP and BLASTX programs (1) via the
National Institute for Biotechnology Information server
(http://www.ncbi.nlm.nih.gov/cgi-bin/blast). Protein secondary
structure predictions were performed with the GORI program
(11) via the ExPASy server
(http://expasy.hcuge.ch/www/tools.html). Pairwise and multiple protein
sequence alignments were made with ALIGN (43) and CLUSTAL W
(39) programs, respectively, at the Baylor College of
Medicine-Human Genome Center server
(http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html). The E. coli database collection ECDC (25) was
accessed via the Internet (http://susi.bio.uni-giessen.de/ecdc.html).
Purification of the reductase component HpaC.
E. coli
DH1 cells harboring plasmid pAJ28 were cultured overnight at 37°C in
2 liters of Luria-Bertani medium containing 0.1 mg of ampicillin per
ml. Cells were harvested by centrifugation, washed, and suspended in
100 ml of 50 mM HEPES buffer (pH 7.8). Cells were broken by passage
through a French press (Aminco Corp.) operated at a pressure of 20,000 lb/in2, and the resulting cell extract was clarified by
centrifugation at 26,000 × g for 30 min. Proteins
contained in the clear supernatant fluid were precipitated with 60%
ammonium sulfate at 4°C. The pellet was recovered by centrifugation
and dialyzed against 50 mM Tris-HCl buffer, pH 8.0 (buffer A). The
soluble protein was loaded on a DEAE-cellulose column (50-ml bed
volume) equilibrated with buffer A. Proteins were eluted at a rate of 1 ml/min with 250 ml of ammonium sulfate in an increasing gradient from 0 to 0.5 M in buffer A with Bio-Rad Econo-System equipment. The fractions showing flavin reductase activity were pooled and loaded onto a
phenyl-Sepharose CL-4B column (12-ml bed volume) equilibrated with
buffer A plus 0.3 M ammonium sulfate. Proteins were eluted at a rate of
0.16 ml/min with 100 ml of a gradient with a decreasing concentration
of ammonium sulfate (0.3 to 0 M) in buffer A. Under these conditions,
the reductase eluted at the end of the gradient after the column was
washed with 20 ml of buffer A. The fractions showing the highest
reductase activity were pooled, concentrated by centrifugation with a
Centricon 10 filter (Amicon) at 11,000 × g for 30 min,
and loaded on a hydroxyapatite-HTP column (5 ml of bed volume)
equilibrated with 10 mM Na-phosphate buffer, pH 7.0. The unbound
protein containing the reductase activity was recovered by washing the
column with 10 ml of the equilibration buffer. Fractions with reductase
activity were concentrated with a Centricon 10 filter and loaded on a
column of Sephadex G-100 (20 by 0.6 cm) that was equilibrated and
eluted with buffer A at a flow rate of 0.2 ml/min. The reductase
activity that was recovered as a sharp peak in the void volume was
stored at 
20°C. The yield and fold purification of HpaC were 40%
and 166, respectively.
Purification of the oxygenase component HpaB.
The native
oxygenase component HpaB was partially purified from extracts of
E. coli DH1(pAJ27) cells by Blue Sepharose columns as
described elsewhere (32). The chimeric CH-HpaB oxygenase was
purified from E. coli TG1(pAJ31) cells by affinity on
DEAE-cellulose through a single chromatographic step as previously
described (35). In short, cells were broken by using a
French press, and the resulting cell extract was clarified by
centrifugation and loaded on a DEAE-cellulose column (10-ml bed volume)
equilibrated with 20 mM sodium phosphate buffer, pH 6.9 (buffer B). The
column was washed with 20 volumes of buffer B containing 1.5 M NaCl. The chimeric protein was eluted with 2 volumes of buffer B containing 1.5 M NaCl plus 140 mM choline. Fractions showing oxygenase activity were pooled, dialyzed against 2 liters of buffer A, and stored at
20°C after addition of 10% glycerol. The yield and fold
purification of CH-HpaB were 47% and 12.5, respectively.
Molecular mass determination.
Fractions containing enzyme
activity were tested for purity by SDS-PAGE (26) with 12.5%
polyacrylamide gels and a molecular mass marker kit for determination
of the subunit molecular mass. Polyacrylamide gels were stained with
Coomassie brilliant blue. The molecular mass of the native protein was
determined by gel filtration analysis on a Superose 12 HR 10/30 column
equilibrated with 50 mM sodium phosphate buffer, pH 8.0, with Gilson
high-performance liquid chromatography (HPLC) equipment. The standards
used to calibrate the column were ferritin (480,000 Da), catalase
(240,000 Da), alcohol dehydrogenase (150,000 Da), bovine serum albumin (67,000 Da), ovalbumin (45,000 Da), and chymotrypsinogen A (25,000 Da).
HPLC analysis of 4-HPA consumption and 3,4-DHPA production.
The production of 3,4-DHPA was analyzed with Gilson HPLC equipment with
a Lichrosphere 5 RP-8 column (150 by 4.6 mm) after a guard column
(mobile phase, 20% methanol-water containing 0.1% [vol/vol]
trifluoracetic acid; flow rate, 1 ml/min). The detection was carried
out spectrophotometrically at 280 nm. Metabolites were identified by
comparison of their retention times with those of pure substances.
Enzyme assays.
Flavin:NAD(P)H reductase activity was
detected by a spectrophotometric assay measuring the disappearance of
the yellow color due to the reduction of FMN by NADH at 450 nm (
= 12,200 M
1 cm1) (18). The assay
cuvette contained 0.06 mM FMN and 5 mM NADH in 50 mM HEPES buffer (pH
7.8), in a final volume of 0.5 ml. After the addition of the enzyme,
the assay was run at 22°C during a controlled period of time. To
determine the enzyme specificity, FAD (450nm = 11,300 M1 cm1) (18), riboflavin
(450nm = 12,200 M1 cm1)
(18), and NADPH were also tested as substrates.
Flavin:NADH oxidase activity was assayed at 22°C by monitoring the
decrease in absorption of NADH at 340 nm ( = 6,220 M1
cm1) (10) in 50 mM HEPES buffer, pH 7.8, containing 0.2 mM NADH and 0.01 mM flavin. Assays were initiated by the
addition of the enzyme. Steady-state kinetic measurements were
performed with a 1-cm light path cuvette in a final volume of 0.5 ml
with a Shimadzu UV-160 spectrophotometer. This assay was used to
determine the Km values for flavins.
NADH:cytochrome
c reductase activity was assayed by
recording the NADH-dependent reduction of cytochrome
c at
550 nm (
=
21,100 M
1 cm
1) (
4)
with 50 mM HEPES buffer, pH 7.8, at 22°C; the reaction
mixture
contained 0.04 mM cytochrome
c, 0.2 mM NADH, and 0.03
mM FMN
or
FAD.
NADH:iron(III) reductase activity was determined with ferrozine as the
iron (II) trap. The reaction was followed by recording
the absorbance
at 562 nm of the ferrozine-iron(II) chelate (
= 28,000 M
1 cm
1) (
10). The assay was
performed at 22°C in 50 mM HEPES buffer,
pH 7.8, containing 0.2 mM
ferric citrate, 1 mM ferrozine, 3 mM
NADH, and 0.02 mM
FMN.
Oxygenase assays were performed at 22°C in 50 mM HEPES buffer, pH
8.0, containing 4 mM 4-HPA, 3 mM NADH, 0.01 mM FAD, 50 mM
glucose, 120 U of catalase/ml, and 0.5 U of glucose dehydrogenase/ml.
The mixture
was gently stirred. Catalase was added to the reaction
mixture to avoid
accumulation of H
2O
2 produced due to
substrate-independent
oxygen consumption. Glucose dehydrogenase was
added to regenerate
the NADH. The reaction was stopped at different
times with 5%
trichloroacetic acid (wt/vol) and the samples were
centrifuged
at 30,000 ×
g for 10 min before the
production of 3,4-DHPA was
analyzed by
HPLC.
Oxygenase activity was also determined by a two-compartment reaction
assay. In this case, a solution (1 ml) carrying the HpaC
reductase (0.6 µg of purified protein) in 50 mM HEPES buffer was
placed inside a
dialysis bag (6 mm in diameter; molecular weight
cutoff, 12 to 14 kDa;
Dialysis SERVA Visking) that was immersed
into a solution (3 ml) of 50 mM HEPES buffer, pH 8.0, containing
4 mM 4-HPA, 3 mM NADH, 0.01 mM FAD,
50 mM glucose, 120 U of catalase/ml,
0.5 U of glucose dehydrogenase/ml,
and the CH-HpaB oxygenase (30
µg of purified protein). The reaction
was performed at 22°C with
shaking, and the resulting 3,4-DHPA was
analyzed by HPLC at different
time intervals from 10 to 300 min. A
control experiment was carried
out under identical conditions but with
the same amount of HpaC
placed outside the dialysis
bag.
N-terminal amino acid sequencing.
The N-terminal amino acid
sequence was determined by Edman degradation with an Applied Biosystems
model 470A gas phase sequencer fitted with an online PTH analytical system.
Protein determination.
Protein was determined by the method
of Bradford (7) with bovine serum albumin as a standard.
| |
RESULTS AND DISCUSSION |
Purification and characterization of the small component (HpaC) of
4-HPA 3-monooxygenase from E. coli W.
We had observed
that the 4-HPA 3-monooxygenase activity of HpaB was significantly
increased after the addition of extracts of E. coli
DH1(pAJ28) cells overexpressing the HpaC protein (32). Moreover, in vitro analyses demonstrated that the hydroxylating activity of HpaB was NADH and FAD dependent (32). Although
the purified HpaB protein did not show the characteristic absorption bands of flavin enzymes, we assumed that the FAD and/or NADH binding sites of the 4-HPA 3-monooxygenase should be located in HpaB, since it
can be specifically eluted by NADH from a Blue Sepharose column,
whereas the HpaC protein did not bind to this matrix (32). Assuming that the behavior of HpaC resembled that of the coupling protein of the 4-HPA 3-hydroxylase from P. putida (2,
3), a similar role was tentatively ascribed to this protein
(32). However, the possibility that the hpaC gene
could encode a reductase instead of a coupling protein was not
envisioned until a similar protein, the ORF6 of the actinorhodin
cluster from Streptomyces coelicolor (hereafter named the
ActVB protein), was shown to behave as a flavin:NADH oxidoreductase
(21). According to this observation, analyses carried out
with extracts of E. coli DH1(pAJ28) revealed the presence of
a high level of flavin:NADH oxidoreductase activity compared with
control extracts of E. coli DH1 cells harboring plasmid
pUC18 (see below). To ascertain that FMN reduction in the presence of
NADH was carried out specifically by the HpaC protein and not by
another enzyme induced in the host cell as a consequence of the
overexpression of the hpaC gene, we decided to purify the
putative HpaC oxidoreductase enzyme.
The purification of flavin:NADH oxidoreductase activity from crude
extracts of
E. coli DH1(pAJ28) cells rendered a protein
of
at least 95% homogeneity, as judged by denaturing gel electrophoresis
(Fig.
1A). The purified enzyme showed an
apparent molecular weight
on SDS-PAGE of 20,000, which was in agreement
with the predicted
molecular mass for the HpaC protein (
32).
Moreover, its N-terminal
amino acid sequence analysis revealed a
sequence, Met-Gln-Leu-Asp-Glu,
which was identical to that deduced from
the nucleotide sequence
of the
hpaC gene (
32).
These findings strongly supported the
assumption that the reductase
activity observed in crude extracts
of
E. coli DH1(pAJ28)
corresponded to that of the HpaC protein.
The purified HpaC protein was
very stable at
20°C, since no significant
loss of activity was
observed during 2 months of storage at this
temperature.
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FIG. 1.
SDS-PAGE analysis of the overproduction and purification
of the two components of the 4-HPA 3-monooxygenase from E. coli W. (A) Lane 1, molecular mass markers; lane 2, soluble
control extract from E. coli DH1(pUC18); lane 3, soluble
crude extract from E. coli TG1(pAJ28); lane 4, purified HpaC
reductase. (B) Lane 1, molecular mass markers; lane 2, soluble control
extract from E. coli TG1(pUC18); lane 3, soluble crude
extract from E. coli TG1(pAJ31); lane 4, purified CH-HpaB
protein. The molecular mass marker proteins are indicated in
kilodaltons.
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Gel permeation chromatography on a Superose 12 HR column of crude
extracts from
E. coli DH1(pAJ28) indicated that HpaC formed
dimers. However, purified HpaC eluted as a high molecular weight
protein, which indicates the formation of soluble aggregates.
A similar
behavior has been also observed with the ActVB reductase
from
S. coelicolor, which is a dimer with a high tendency to form
aggregates when purified (
21).
Biochemical properties of the HpaC oxidoreductase.
The
purified HpaC oxidoreductase was colorless, and the UV-visible spectrum
showed no evidence for any chromogenic cofactor (data not shown). The
reductase activity of HpaC depended on both NADH and flavin being added
to the assay. No requirement for any other cofactors was apparent. In
particular, these experiments showed that 4-HPA had no influence in
this reaction. The most effective substrates were NADH and FMN, but FAD
and riboflavin could also be turned over by the enzyme with similar
Km values (see below). When the reductase assay
was performed without shaking, FMN was reduced completely by an excess
of NADH, and the absorption band at 450 nm corresponding to FMN was
completely bleached. After shaking, reduced flavin mononucleotide
(FMNH2) was recycled by reaction with oxygen to form
H2O2, and the absorption at 450 nm returned.
When FMN was added in a 200-fold molar excess of the HpaC protein, it
became completely reduced (data not shown), suggesting that the flavin
dissociated from the protein and behaved as a true substrate rather
than as a tightly bound cofactor.
The HpaC reductase showed optimal activity at pH 7.0, but it maintained
more than 80% of activity between pH values of 6.5
to 8. The
Km values for NADH and different flavins (Table
1)
were similar to those observed for
other flavin reductases, that
is, enzymes that generate free reduced
flavins (
10,
18,
21,
38). It is worth noting the high
Km values for FMN when compared
to the
Km values in the nanomolar range that have been
determined
for other monooxygenases, an observation that reinforces the
idea
that FMN acts as a substrate rather than as a prosthetic group.
The specific activity of HpaC on different flavins using NADH
as an
electron donor (Table
1) was similar to that reported for
the SnaC
reductase (
38) but 10 times higher than that reported
for
ActVB reductase (
21), the other two HpaC-like reductases
(see below) that have been purified so far. Other flavin reductases
that do not show sequence similarity to HpaC, like the Fre reductase
from
E. coli (
10) and the major flavin reductase
(FRase I) from
Vibrio fischeri (
18), also showed
specific activities of around
100 µmol min
1
mg
1. Although the HpaC enzyme can also use NADPH as a
substrate,
its specific activities on FMN, FAD, and riboflavin were
more
than 2 orders of magnitude lower than those observed in the
presence
of NADH (Table
1). This behavior appears to be typical of
flavin
reductases that do not contain a flavin as a prosthetic group,
since they reduce FMN, FAD, and riboflavin with similar efficiencies
but present a higher selectivity for NADH or NADPH (
10,
18,
21,
38). The low specificity could be ascribed to the fact
that in
these reductases, the flavin behaves as a real substrate
and not as a
tightly bound prosthetic group, as is the case in
the majority of
flavin enzymes (
12).
The HpaC oxidoreductase can also reduce cytochrome
c and
iron(III) at a high velocity (Table
1). Although cytochrome
c reductase
activity has also been reported for the
reductase components of
pyrrole-2-carboxylate monooxygenase
(
4) and the chlorophenol
4-monooxygenase (
16),
two putative members of the TC-FDM family
(see below), ferric reductase
activity had not been detected so
far in any other reductase component
of this family of monooxygenases.
It is worth noting that the ferric
and cytochrome
c reductase
activities had been considered a
peculiar characteristic of Fre
reductase from
E. coli
(
10) or of the major flavin reductase
(FRase I) from
V. fischeri (
18). Therefore, in all these cases,
the electron transfer from NADH to other electron acceptors appears
to
be mediated through FMNH
2 generated by a
reductase.
Analysis of the oxygenase-reductase interactions in the 4-HPA
3-monooxygenase.
Although we have observed that HpaC was able to
produce FMNH2 and reduced flavin adenine dinucleotide
(FADH2) in vitro in the absence of 4-HPA, it was necessary
to investigate whether such activity could be affected by the presence
of the oxygenase component HpaB. In spite of the fact that the HpaB
protein had been purified, it lost most of its original activity due to
its low stability and to the time consumed by the complex purification procedure (32). When we tried to purify the HpaB protein by a single Blue Sepharose chromatography, the partially purified enzyme
represented about 17% of the total protein, as determined by SDS-PAGE,
and showed an activity of 140 nmol min1 mg1
in the presence of saturating concentrations of NADH and HpaC reductase
(data not shown). Although this HpaB preparation presented a high level
of activity, the possibility that some host reductase(s) could be
retained in the Blue Sepharose column and thereafter coeluted with HpaB
could not be ruled out. In fact, we have detected a low FAD-dependent
reductase activity (5 nmol min1 mg1) in
HpaB preparations. Therefore, to purify the HpaB enzyme by a faster and
more selective procedure, we constructed a chimeric tagged HpaB protein
(CH-HpaB) by fusing the choline-binding domain of the major autolysin
of Streptococcus pneumoniae (35) to the N
terminus of HpaB (Fig. 2). The CH-HpaB
protein can be purified free of reductase-contaminating activities in a
single step by affinity chromatography with DEAE-cellulose, a choline
analogue-containing matrix (35). E. coli TG1
cells harboring plasmid pAJ31 produced large amounts of the CH-HpaB
fusion both as soluble and insoluble (inclusion bodies) protein. The
soluble protein was recovered by centrifugation and loaded on a
DEAE-cellulose column. Figure 1B shows that the CH-HpaB protein eluted
in the choline fraction is nearly pure. Interestingly, the purified
CH-HpaB protein did not show a contaminating flavin reductase activity
(data not shown) but presented a high level of oxygenase activity in
the presence of saturating concentrations of NADH and HpaC reductase
(see below).
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FIG. 2.
Schematic representation of the construction of the gene
encoding the CH-HpaB fusion protein. Abbreviations: Apr,
ampicillin resistance; Cmr, chloramphenicol resistance; E,
EcoRI; H, HindIII; S, SalI;
Plac, lac promoter;
Plpp, lpp promoter; RBS, ribosome
binding site. Amino acids are indicated by their standard single letter
codes.
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To investigate the oxygenase activity of CH-HpaB in the absence or
presence of HpaC, we determined the rate of synthesis of
3,4-DHPA from
4-HPA in vitro. Since the reaction must be carried
out under aerobic
conditions, we included an NADH-regenerating
system and catalase in the
assay to keep the concentration of
NADH constant and to avoid the
accumulation of H
2O
2, respectively.
Interestingly, as we have avoided the contamination of the purified
CH-HpaB protein with any flavin reductase, the chimeric enzyme
was
unable to hydroxylate 4-HPA in the absence of HpaC reductase.
As
expected, the addition of HpaC to CH-HpaB reaction mixtures
(1:5 molar
ratio) led to the production of 3,4-DHPA with activity
levels of 460 nmol min
1 mg
1. This hydroxylating activity
was NADH and FAD dependent, and
neither FMN nor riboflavin could
replace FAD in the reaction.
This result is in agreement with the
previous finding that hydroxylation
of 4-HPA was stimulated by the
addition of FAD to the assay (
32).
Although we were able to
detect a very low level of hydroxylating
activity when NADH was
replaced by NADPH (data not shown), most
probably this observation does
not have any physiological relevance.
The specific activity of the
chimeric CH-HpaB enzyme was close
to the theoretical value (800 nmol
min
1 mg
1) that can be deduced from the
activity of the wild-type HpaB
enzyme partially purified on a Blue
Sepharose column and the SDS-PAGE
densitometric determination of the
HpaB content in this preparation
(see above). Taking into account all
these results, it can be
concluded that the fusion of the
choline-binding domain to the
N terminus of HpaB does not significantly
affect its enzymatic
activity but facilitates a rapid purification of
the protein free
from contaminant reductase activities. The use of this
choline-binding
domain offers, therefore, a suitable alternative for
investigating
the overexpression and easy purification of other
oxygenases.
The observation that CH-HpaB activity was absolutely dependent on the
presence of HpaC supports the hypothesis that both components
are
required for 4-HPA hydroxylation. Therefore, the low level
of
HpaC-independent oxygenase activity (0.5 nmol min
1
mg
1) detected with the native HpaB purified by the Blue
Sepharose
method should be ascribed to a contaminant flavin:NADH
reductase
from the host (see above) that generates the
FADH
2 required for
4-HPA
hydroxylation.
To determine whether a physical interaction between the two components
of the 4-HPA 3-monooxygenase is indispensable to catalyze
the
hydroxylation of 4-HPA, the HpaB and HpaC components were
placed in two
different compartments separated by a membrane permeable
to compounds
smaller than 14 kDa (see Materials and Methods).
By this
two-compartment reaction assay, 4-HPA was efficiently
transformed into
3,4-DHPA at a rate of 138 nmol min
1 mg
1.
This value is in the same order of magnitude of that obtained
in a
control experiment (345 nmol min
1 mg
1)
carried out under the same conditions but with the reductase
and
oxygenase components placed in the same compartment. This
result
indicated that a physical interaction between HpaB and
HpaC is not
required for the hydroxylation of 4-HPA, although
we cannot discard the
idea that a direct interaction between HpaB
and HpaC could enhance the
hydroxylation reaction. Interestingly,
the presence or absence of the
CH-HpaB component did not affect
the levels of FMN reduction by the
HpaC reductase. All these data
suggest that HpaC reduces FAD to
FADH
2, which then dissociates
from the enzyme and diffuses
to the medium, where it is captured
by HpaB to catalyze the
hydroxylation of 4-HPA. Since the HpaB
oxygenase component does not
require a direct interaction with
the HpaC oxidoreductase to
hydroxylate 4-HPA, any flavin reductase
present in the host cell that
is able to release FADH
2 into the
cytoplasm would supplant
the role of HpaC. This finding explains
the puzzling result observed in
E. coli DH1(pAJ271), a strain
that expressed the oxygenase
HpaB component alone but showed a
significant level of 4-HPA
hydroxylating activity (
32).
Structural and evolutionary analyses of the TC-FDM family.
Table 2 shows a compilation of the
monooxygenases described so far that might be tentatively classified as
members of the TC-FDM family. This family can be defined according to
the following properties. (i) The reductase and the oxygenase
components of the monooxygenase are encoded by two different genes.
(ii) The reductase component uses NAD(P)H to catalyze the reduction of a flavin that diffuses to the oxygenase component for oxidation of the
substrate (aromatics or nonaromatic compounds) by molecular oxygen.
(iii) Both components are not flavoproteins, i.e., they do not contain
any flavin prosthetic group and lack typical ferredoxin and/or
flavin:NAD(P)H binding motifs. Interestingly, no three-dimensional structure is known for any of these proteins. It is worth noting that
the pristinamycin IIA synthase was not included in Table 2
because although SnaC reductase shows a significant similarity with the
HpaC protein, the oxygenase component is certainly an 
heterodimer (6). Similarly, in spite of the fact that
bacterial luciferase is probably the best-understood system in which
the oxygenase component uses the FMNH2 produced by a
reductase component (18), it was not included in Table 2
because its oxygenase component is also an heterodimer.
Furthermore, none of the luciferase components show a significant
similarity with the reductase and oxygenase components of any member of
the TC-FDM family. Finally, the two-component 4-HPA hydroxylase of
P. putida was also not included in Table 2 since,
apparently, it does not involve a flavin reductase component (2,
3).
The oxygenase components of the members of the TC-FDM family show
marked differences in their primary structures, which might
reflect the
fact that the substrate specificity of these enzymes
resides in these
components (
20). Interestingly, amino acid
sequence
comparisons among the oxygenase components of several
aromatic
hydroxylases of the TC-FDM family have revealed the existence
of a
conserved region (Fig.
3). Remarkably, a
54% identity has
been observed between the HpaB oxygenase component of
4-HPA 3-monooxygenase
from
E. coli W and the phenol
hydroxylase (PheA) from
Bacillus thermoleovorans
(
9). This high amino acid sequence identity
agrees with the
observation that phenol is also a satisfactory
substrate for HpaB
(
31,
32), suggesting that both enzymes
might have evolved
from a common ancestor able to hydroxylate
phenol derivatives. Although
no biochemical data on the cofactor
requirements of PheA activity are
available (
9), the significant
similarity observed between
HpaB and PheA suggests that the latter
does not contain a
flavin:NAD(P)H reductase center, and therefore,
it is likely to require
an independent reductase enzyme which
could provide this activity. On
the other hand, it is worth noting
that the central region of the
HpaB-like oxygenases displays a
significant similarity with a central
region of the medium-chain
acyl-coenzyme A dehydrogenases (data not
shown) that is in close
contact with the flavin nucleotide
(
23). Nevertheless, whether
the central region of HpaB-like
oxygenases has a role in the interaction
with the reduced flavin
provided by the cognate reductase component
remains to be checked.
View larger version (38K):
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|
FIG. 3.
Multiple sequence alignment of the oxygenase components
of several members of the TC-FDM family. Numbers in parentheses
indicate the position of the residues in the complete amino acid
sequence of the protein. A consensus sequence was deduced for positions
where the residues were identical in more than half of the sequences.
AF-HpaA1, putative 4-HPA 3-monooxygenase from Archaeoglobus
fulgidus (AE001081); AF-HpaA2, putative 4-HPA 3-monooxygenase from
A. fulgidus (AE001043); AF-HpaA3, putative 4-HPA
3-monooxygenase from A. fulgidus (AE001032); PA-PvcC
putative hydroxylase of pyoverdine chromophore biosynthesis in
Pseudomonas aeruginosa (AF002222); PL-HpaB, putative 4-HPA
3-monooxygenase from Pseudomonas luminescens (AF021839);
KP-HpaA, 4-HPA 3-monooxygenase from Klebsiella pneumoniae
(L41068); EC-HpaB, 4-HPA 3-monooxygenase from E. coli
(Z29081); SD-HpaB, putative 4-HPA 3-monooxygenase from S. dublin (AF144422); BT-PheA, phenol hydroxylase from B. thermoleovorans (AF031325); BS-Yoal, putative 4-HPA
3-monooxygenase from Bacillus subtilis (Z99114); BP-HadA;
chlorophenol 4-hydroxylase from B. pickettii (D86544);
BC-TftD, chlorophenol 4-hydroxylase from B. cepacia
(U83405); RE-DszC, dibenzothiophene monooxygenase from R. erythropolis (L37363); PP-MsuC, putative monooxygenase from
P. aeruginosa (AF026067).
|
|
In contrast with the oxygenase components, we have observed an extended
similarity among the reductase components of the TC-FDM
proteins (Fig.
4). The only exceptions were the MsuE and
the related
SsuE reductases (Table
2) that lack similarity with any
other
described reductases (
22). The similarities among the
reductases
correlate with the fact that all of them use the same
substrates,
i.e., FAD-FMN and NAD(P)H. Although most of the HpaC-like
reductase
components are colorless proteins, suggesting that flavin is
not
tightly bound to the enzyme, there are some exceptions, such as
the
NtaB reductase component of the nitrilotriacetate monooxygenase
from
Chelatobacter heintzii that shows a typical FMN spectrum
although the flavin is not strongly bound to the enzyme
(
40).
A similar behavior was also observed with the cB'
reductase component
of the EDTA-monooxygenase (
44).
View larger version (65K):
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|
FIG. 4.
Multiple sequence alignment of HpaC-like proteins. A
comparison of the amino acid sequences of HpaC and other proteins of
the databases that present a significant similarity is shown. Numbers
in parentheses indicate the position of the residues in the complete
amino acid sequence of the protein. A consensus sequence was deduced
for positions where the residues were identical in more than half of
the sequences. SC-ActVB, flavin reductase involved in the biosynthesis
of actinorhodin in S. coelicolor (X58833); SR-FrnH, putative
flavin reductase involved in frenolicin biosynthesis in
Streptomyces roseofulvus (AF058302); SV-Orf34, putative
flavin reductase involved in granaticin biosynthesis in
Streptomyces violaceoruber (AJ011500); SV-VlmR, putative
flavin reductase involved in valanimycin biosynthesis in
Streptomyces viridifaciens (U93606); SP-SnaC, flavin
reductase involved in pristimamycin IIA biosynthesis in
Streptomyces pristinaespiralis (P54994); SA-Orf, putative
reductase involved in chlortetracycline biosynthesis in
Streptomyces aureofaciens (D38215); CH-NtaB, flavin
reductase component of the nitrilotriacetate monooxygenase from
C. heintzii (U39411); CH-NmoB, flavin reductase component of
the nitrilotriacetate monooxygenase from C. heintzii
(L49438); RE-Bph61, putative reductase involved in the metabolism of
biphenyl derivatives in R. erythropolis (D88018); MT-14c,
putative reductase from Mycobacterium tuberculosis (Z92774);
PF-StyB, flavin reductase component of the styrene monooxygenase from
Pseudomonas fluorescens (Z92524); PY-StyB, flavin reductase
component of the styrene monooxygenase from Pseudomonas sp.
strain Y2 (AJ000330); PS-StyB flavin reductase component of the styrene
monooxygenase from Pseudomonas sp. strain VLB120 (AF031161);
MT-11, putative reductase from M. tuberculosis (AL021929);
ML-23, putative reductase from Mycobacterium leprae
(AL022486); MT-25c, putative reductase from M. tuberculosis
(Z84498); RE-DszD, flavin reductase involved in dibenzothiophene
desulfurization from R. erythropolis (AF048979); PL-HpaC,
HpaC-like reductase from P. luminescens (AF021838); KP-HpaH,
putative reductase component of 4-HPA 3-monooxygenase from K. pneumoniae (L41068); EC-HpaC, reductase component of 4-HPA
3-monooxygenase from E. coli (Z29081); EC-F152, putative
HpaC-like reductase from E. coli (AE000202); SD-HpaC,
putative HpaC reductase from S. dublin (AF144422); BC-TftC,
reductase component of chlorophenol 4-hydroxylase from B. cepacia (U83405); AF-HpaCL, putative reductase from A. fulgidus (AE001047); SS-HpaC-1, HpaC-like protein from
Synechococcus sp. (L19521); SS-HpaC-2, HpaC-like protein
from Synechococcus sp. (D64000); SS-F594, flavoprotein from
Synechocystis sp. (D90900); SS-F578 flavoprotein from
Synechocystis sp. (M96929); SS-F597 flavoprotein from
Synechocystis sp. (D90914); SS-F573, flavoprotein from
Synechocystis sp. (D64003).
|
|
Organisms have evolved a great variety of enzymes that catalyze the
reduction of flavins by NAD(P)H. These flavin:NAD(P)H
oxidoreductases
can be classified within several families and
subfamilies according to
their sequence similarities and biochemical
properties. One of these
families is constituted by the flavoprotein
reductases that contain a
tightly bound flavin as a prosthetic
group, e.g., the Frp reductase
from
Vibrio harveyi (
27) and
the sulfite
reductase from
E. coli (
8), which are
representative
members of two different subfamilies. Another family is
represented
by those enzymes that do not contain a flavin as a
prosthetic
group and thus cannot be considered flavoproteins. Instead,
they
use flavins as substrates, with a rather broad substrate
specificity.
Based on sequence comparisons, at least two subfamilies
were identified,
one constituted by the Fre reductase from
E. coli as well as the
Fre-like and LuxG reductases of luminous
bacteria (
17), and
the other constituted by the FRase I from
V. fischeri (
18).
Interestingly, amino acid
sequence comparison analyses revealed
that the HpaC-like reductases are
not similar to other nonflavoprotein
flavin reductases described so far
and therefore they appear to
constitute a novel
subfamily.
Multiple sequence alignment of flavin reductases of the HpaC subfamily
revealed several conserved residues (Fig.
4). Thus,
a residue of Ser
(Thr or Cys) located before a pair of conserved
proline residues is
highly conserved at the N termini of the proteins
(Fig.
4). Since Gly,
Asp, and His residues at the C-terminal region
of Fre are involved in
NAD(P)H binding (
17), it is tempting
to assume that the
highly conserved GDH motif found in the C-terminal
regions of members
of the HpaC family could play a role in NAD(P)H
interaction.
Only two flavin reductases have been described so far in
E. coli, the Fre reductase (
17) and the sulfite reductase
(
8).
However, the findings reported here demonstrate the
existence
in
E. coli W of another highly active reductase,
the HpaC reductase,
that can be considered a prototype of a new
subfamily of nonflavoprotein
flavin reductases. The existence of
several reductases capable
of producing free reduced flavins in a
microorganism, and the
apparent functional interchangeability between
them (
29) poses
some questions. A potential adaptive
significance of this redundancy
is to provide a readily available
backup if an enzyme is lost
by a mutational event. For instance,
although the Fre reductase
of
E. coli appears to provide
reduced flavins for specific purposes,
it has been shown that the
sulfite reductase can replace its activity
in Fre-deficient mutants
(
8). Similarly, as it has been pointed
out above, the
existence of Fre or other flavin reductases could
explain the residual
4-HPA 3-monooxygenase activity observed in
E. coli K-12
cells expressing only the HpaB oxygenase component
(
32).
Nevertheless, since the amount of FADH
2 provided by the
host reductases is not enough to achieve an optimal 4-HPA
3-monooxygenase
activity, the acquisition of the
hpaC
reductase gene cotranscribed
(coregulated) with the
hpaB
oxygenase gene might represent an
evolutionary advantage for the
development of a highly efficient
4-HPA catabolic
pathway.
The genes encoding the two components of the enzymes of the TC-FDM
family are located in the same operon or can be found very
close in the
chromosome (
32), sometimes divergently oriented
(
24). Such an arrangement could favor a coordinated
regulation
of both genes and might facilitate the horizontal transfer
of
the monooxygenase activity. Nevertheless, there are several
exceptions
to this arrangement and thus the
dszD gene
encoding the reductase
component involved in the S oxidation of
dibenzothiophene is located
on the genome far from the genes encoding
the DszA and DszC oxygenase
components (
45). This genetic
arrangement and the observed functional
exchangeability among the
flavin reductases (
29) suggest that
the genes encoding both
components of the enzymes of the TC-FDM
family might have evolved
independently but frequently became
physically associated in the
genome.
A further step in the evolution of the HpaC-like reductases can be
inferred from their amino acid sequence similarities to
the C-terminal
extension of four A-type flavoproteins from
Synechocystis (Fig.
4) (
42). It was suggested that these flavoproteins,
which
bind FAD and FMN at the same time in equimolecular amounts, might
have evolved by the fusion of two flavin-binding domains located
in the
N- and C-terminus regions of the protein, showing different
activities
and functions (
42). The existence of a HpaC-like
FMN binding
domain in A-type flavoproteins suggests that a fusion
between the
reductase and oxygenase components of the enzymes
of the TC-FDM family
might already exist in nature or, at least,
it would be feasible to
design in vitro such monocomponent monooxygenase
by protein engineering
(unpublished data). An evolutionary mechanism
similar to that proposed
here has been postulated to explain the
origin of phthalate dioxygenase
reductase (
17).
The comparative analyses presented here also provide valuable
information for ascribing functions to several still-unclassified
genes
found in recently sequenced genomes. Some putative HpaC-like
flavin
reductases that have been found in different microorganisms
are
included in the multisequence alignment shown in Fig.
4. Interestingly,
a comparative analysis of the
E. coli genome has revealed
two
open reading frames (ORFs),
orf152 and
orf196
(
AE000202), that
may encode two proteins showing 41 and 53% amino acid
sequence
identity with HpaC and HadB, respectively.
orf152
is located at
23 min in the
E. coli K-12 linkage map,
immediately downstream
of
orf196 and within the same
putative transcription unit that
also contains five additional ORFs of
unknown function, which
are oriented opposite an ORF encoding a
putative regulatory protein
(
25). Since one of these ORFs
(
orf382) displays a significant
similarity with the DszA
monooxygenase from
Rhodococcus erythropolis (Table
2), it is
reasonable to assume that this
E. coli gene
cluster might
encode an oxygenolytic pathway whose physiological
role is still
unknown. Interestingly, in the
Salmonella dublin genome
there are two contiguous genes,
hpaB and
hpaC
(
AF144422),
whose putative products share 92 and 79% identity with
HpaB and
HpaC from
E. coli, respectively, thus suggesting
the existence
of a 4-HPA 3-monooxygenase of the TC-FDM family in this
enteric
bacteria.
It is surprising that whereas the HadA oxygenase component of the
chlorophenol 4-hydroxylase from
Burkholderia pickettii is
homologous to the TftD oxygenase component of the chlorophenol
4-hydroxylase from
Burkholderia cepacia (Fig.
3), the HadB
component
did not show any similarity with the corresponding TftC
reductase
or with any other HpaC-like flavin reductases. In fact, HadB
shows
significant similarity only with the
H
2O
2-forming NADH oxidase
from
Thermus
thermophilus HB8 (
28). Interestingly, we have
identified
just at the 5' end of the
hadAB operon of
B. pickettii (
36),
a partially sequenced ORF that
might form part of the same operon
and that encodes a protein showing
23% amino acid identity with
the HpaC reductase (data not shown).
Whether the protein encoded
by this ORF could be the real reductase
component of the chlorophenol
4-hydroxylase from
B. pickettii instead of the proposed HadB protein
(Table
2) requires
further
research.
In summary, the results presented above demonstrate that HpaC is a
flavin:NADH oxidoreductase involved in the hydroxylation
of 4-HPA by
the HpaB oxygenase component of the 4-HPA 3-monooxygenase
and therefore
allow us to complete the functional characterization
of all the genes
of the 4-HPA catabolic cluster in
E. coli W (
31).
Since the 4-HPA 3-monooxygenase from
E. coli W was the first
member
of the new TC-FDM family whose primary structure was elucidated
(
32), this enzyme can be considered the archetype of this
family,
with the HpaC reductase being, in turn, the prototype of a new
subfamily of flavin
reductases.
| |
ACKNOWLEDGMENTS |
We thank J. Varela, A. Díaz, S. Carbajo, and G. Porras
for their help with protein and DNA sequencing. We are indebted to M. Carrasco and E. Cano for their technical assistance. M. A. Prieto
was a recipient of a Contrato de Incorporación de Doctores del
Ministerio de Educación y Cultura.
This work was supported by grant AMB97-0630-C02-02 from the CICYT.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Molecular Microbiology, Centro de Investigaciones Biológicas,
Consejo Superior de Investigaciones Científicas,
Velázquez 144, 28006 Madrid, Spain. Phone: 34-91-5611800. Fax:
34-91-5627518. E-mail: JLGARCIA{at}CIB.CSIC.ES.
| |
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Journal of Bacteriology, February 2000, p. 627-636, Vol. 182, No. 3
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
