(Received for publication, July 22, 1996, and in revised form, October 24, 1996)
From the Department of Biochemistry and Molecular Biology, College of Medicine and H. Lee Moffitt Cancer Center and Research Institute, University of South Florida, Tampa, Florida 33612
Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 µmol of biotin sulfoxide reduced per min/nmol of enzyme and Km values of 29 and 15 µM for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.
Biotin sulfoxide (BSO)1 reductase, which catalyzes the reduction of d-biotin d-sulfoxide to d-biotin according to the following scheme,
![]() |
(Eq. 1) |
BSO reductase has been partially purified from Escherichia
coli and demonstrated to be a soluble protein that requires an unidentified form of the Mo cofactor (Rajagopalan and Johnson, 1992)
and several accessory proteins for activity (del Campillo-Campbell and
Campbell, 1982
). These accessory proteins include a small, heat stable,
thioredoxin-like protein moiety, referred to as
protein-(SH)2 and which functions as a source of reducing
equivalents and an unidentified flavoprotein (del Campillo-Campbell
et al., 1979
). The extensive characterization of BSO
reductase has been limited by the low natural abundance of the protein,
its constitutive expression, and the requirement for auxiliary proteins
for activity coupled with the difficulty of its detection in the
absence of specific antibodies. While the E. coli BSO
reductase bisC structural gene has been cloned and
sequenced, the enzyme has not been produced using heterologous
expression systems.
Biotin sulfoxide reductase, either from E. coli or
Rhodobacter sphaeroides (Pollock and Barber, 1995), together
with R. sphaeroides Me2SO reductase (Barber
et al., 1995
) and E. coli trimethylamine N-oxide reductase (Mejean et al., 1994
) are
unique members of the small class of metalloenzymes that require Mo for
functionality as their sole prosthetic group. Previous attempts to
develop E. coli-based heterologous expression systems for
this group of MGD-containing enzymes has been limited to R. sphaeroides Me2SO reductase, where the enzyme was
produced in an insoluble and inactive form (Hilton and Rajagopalan,
1996a
).
We have heterologously expressed the R. sphaeroides BSO
reductase as a GST fusion protein (Smith and Johnson, 1988)
facilitating its isolation as a homogeneous, functional enzyme that
contains MGD as its sole prosthetic group. Initial rate kinetic studies have demonstrated that the enzyme can use a variety of substrates, in
addition to BSO and have established for the first time that the enzyme
can also directly use NADPH as an efficient electron donor.
Chemicals, Enzymes, and Reagents
Restriction enzymes and Factor Xa protease were purchased from
Promega (Madison, WI), U. S. Biochemical Corp., and New England Biolabs
(Beverly, MA). Media for bacterial growth was purchased from Difco, and
aprotinin, nucleotide pyrophosphatase (type III), antibiotics, biotin,
thiamine hydrochloride, methyl viologen, benzyl viologen,
p-dimethylaminocinnamaldehyde, reduced glutathione, reduced
thioredoxin (Spirulina sp.) and basic buffer chemicals were
purchased from Sigma. Agarose, urea, SDS, acrylamide
and bisacrylamide, protein molecular weight markers, as well as protein assay solution, were purchased from Bio-Rad.
Isopropylthio--galactoside was purchased from Research Products
International Corp. (Mt. Prospect, IL) and ProBlottTM nylon
membrane was purchased from Applied Biosystems. d-Biotin d-sulfoxide was prepared as described by Pollock and Barber
(1995)
.
Bacterial Strains
E. coli JM109 was purchased from Promega. The mutant
strain of E. coli Mu29 (E. coli
Mu29 is
bio
, bis
,
thi
, strr) was a generous
gift from Dr. Allan Campbell (Stanford University). R. sphaeroides f. sp. denitrificans IL 106 were the
generous gift of Dr. T. Satoh (Hiroshima University).
Vectors
pUC19 was purchased from Boehringer Mannheim and the GST fusion protein expression vector, pGEX-5X-2, was purchased from Pharmacia Biotech Inc.
GST-BSO Reductase Expression Vector Construction
The entire 2.4-kilobase BSO reductase coding region was excised
from the pUC19 construct (Pollock and Barber, 1995) using EcoRI and SalI restriction enzymes and cloned into the
EcoRI and SalI sites of the pGEX-5X-2 expression
vector. DNA sequencing was performed on this construct to confirm the
proper junction sites and reading frame for the BSO reductase gene
using the Sequenase Version 2 (U. S. Biochemical Corp.) sequencing kit
and [
-35S]dATP.
Protein Expression and Purification
Overnight cultures of E. coli JM109 transformed with
either the pGEX-5X-2 vector containing the BSO reductase sequence, or the vector alone as a control, were diluted 1/100 in LB supplemented with ampicillin (100 µg/ml final concentration) and sodium molybdate (1 mM final concentration) and grown at 37 °C to an
OD600 of 0.8. Isopropylthio--galactoside (0.1 mM final concentration) was added to induce protein
expression and the cells were grown for an additional 4 h at
24 ° C. The cells were harvested by centrifugation, resuspended in
phosphate-buffered saline supplemented with dithiothreitol (10 mM), sodium molybdate (1 mM), aprotinin (0.1 mg/ml), EDTA (1 mM), and phenylmethylsulfonyl fluoride (0.1 mM), and sonicated on ice. The sonicated cells were
centrifuged at 30,000 × g for 30 min and the
supernatant chromatographed on a glutathione-agarose affinity column
(8-ml matrix volume) pre-equilibrated with phosphate-buffered saline.
Following several washes with phosphate-buffered saline, the bound
fusion protein was eluted with 50 mM Tris-HCl buffer, containing 10 mM GSSH, pH 8. Fractions exhibiting MV:BSOR
activity were concentrated by pressure filtration (PM-10, Amicon) and
stored in liquid nitrogen. Following cleavage with Factor Xa, the
affinity-purified BSO reductase complex was re-chromatographed using
glutathione-agarose to remove the GST portion of the fusion protein.
Further purification of the BSO reductase to separate the enzyme from
auxiliary proteins was achieved utilizing fast protein liquid
chromatography gel filtration on a Superose 12 column (1 × 30 cm,
Pharmacia) in 50 mM Tris-HCl buffer, pH 8, and anion
exchange on a Mono-Q column (0.5 × 5 cm) utilizing a salt
gradient from 0 to 0.5 M NaCl.
Untransformed E. coli JM 109 and Mu29 cells were grown in
LB to an OD600 of 2.0, respectively. Cells were harvested
and disrupted using identical methods to those described for the
transformed cells and the supernatants assayed for both NADPH:BSOR and
BSO-independent NADPH-oxidation activities, prior to and following
dialysis, in 50 mM Tris, pH 8. R. sphaeroides
cells were grown either aerobically in LB or anaerobically in the
presence of 0.2% nitrate to an OD600 of 2.5, harvested,
and processed as described for the E. coli cells. R. sphaeroides Me2SO reductase was isolated as described previously (Barber et al., 1995
).
Factor Xa Proteolysis
Protein samples were subjected to cleavage by Factor Xa (1% w/w) in 50 mM Tris-HCl buffer, containing 150 mM NaCl and 1 mM CaCl2, pH 8, at 16° C for 16 h.
Protein Analysis
BSO reductase purity was assessed by both SDS-PAGE and
reverse-phase HPLC analysis. Enzyme samples (1-10 µg of total
protein) were analyzed using either 10 or 12.5% SDS-PAGE gels
(Laemmli, 1970) stained with Coomassie Blue. For reverse-phase HPLC
analysis, BSO reductase (30 µg) was mixed with an equal volume of 8 M guanidine hydrochloride, acidified with 0.5% acetic
acid, and chromatographed on a C4 column (4.6 × 30 mm) using a
linear gradient of trifluoroacetic acid (0.1%) to trifluoroacetic acid
(0.1%), acetonitrile (70%).
Molybdenum Cofactor Analysis
Samples of BSO reductase and Me2SO reductase were denatured with 1% SDS for 12 h followed by boiling for 20 min. The SDS was removed by precipitation with KCl (0.250 M), the samples centrifuged at 20,000 × g for 5 min, and the denatured protein separated from the cofactor utilizing ultrafiltration spin columns ("ultrafree-MC" 5,000 MW cut-off, Millipore Corp.). The cofactor-containing solution was assayed for protein and its UV visible and fluorescence spectra recorded. For nucleotide pyrophosphatase cleavage, MgCl2, and lyophilized pyrophosphatase were added to the cofactor-containing solution which was incubated at 37 °C for 15 min following which the cofactor was separated from the pyrophosphatase by ultrafiltration.
Protein Sequencing
NH2-terminal sequencing was performed as described
by Pollock and Barber (1995). For the generation and sequencing of
internal peptides, protein samples were separated by SDS-PAGE,
transbloted onto nylon membranes, and subjected to in situ
proteolytic cleavage. Excised membrane sections were first blocked with
polyvinyl pyrrolidone (1% in methanol) for 30 min, extensively washed
with water and immersed in 100 µl of protease digestion buffer (0.1 M Tris-HCl, pH 8, 1% Triton X-100 and 10% acetonitrile).
Endoproteinase Lys-C (1 µg) was added, and digestion continued at
37 °C overnight. Following proteolysis, the peptide-containing
solution was denatured with guanidine hydrochloride, acidified with
acetic acid, and individual peptides separated by reverse-phase HPLC
(Vydac C18 column, 4.6 × 250 mm) as described by Neame and Barber
(1989)
.
Spectroscopic Analysis
UV-visible spectra were obtained using a Shimadzu Scientific
Inst. Inc. (Columbia MD) UV2101PC spectrophotometer and fluorescence spectra were obtained using a JASCO (Easton, MD) FP770
spectrofluorimeter. UV CD spectra were obtained using a JASCO J710
spectropolarimeter as described by Trimboli et al.
(1996).
Enzyme Activities
Biotin Analysis Using Reverse-phase HPLCThe conversion of
BSO to biotin was performed using MES buffer, pH 6 (140 µl of 114 mM), BSO (50 µl of 5 mg/ml), MV (500 µl of 100 mM in 50 mM Tris buffer, pH 7.5, reduced with
H2 and Pt-asbestos) and homogeneous BSO reductase (30 µl of
0.5 µg/µl). The buffer solution containing BSO was made anaerobic
and extensively bubbled with argon. MV
was added followed by
the BSO reductase and the reaction allowed to proceed anaerobically
until the MV
had been depleted (about 5-10 min, final
solution pH of 8). The reaction mixture was filtered (5,000 Mr cut-off ultrafiltration spin column) to
separate the protein and reaction products and the latter subsequently
analyzed by reverse-phase HPLC (Vydac C18 column, 4.6 × 250 mm)
using a linear gradient of trifluoroacetic acid (0.05%, pH 2.5) to
trifluoroacetic acid (0.05%, pH 2.5), acetonitrile (70:30, v/v) at a
flow rate of 1 ml/min. Control reactions contained MV
and
buffer; BSO, MV
and buffer; biotin (50 µl of 5 mg/ml),
MV
, and buffer; BSO, MV
, heat-inactivated BSO
reductase, and buffer, respectively.
The disk microbiological assay
was performed using a modified version of that previously described by
Pollock and Barber (1995). E. coli
Mu29
(bio
, bis
,
thi
), which cannot grow in the absence of
biotin or in the presence of BSO and absence of biotin, was deposited
onto minimal glucose plates supplemented with tetrazolium (0.0116%),
thi (10 µg/ml), and str (75 µg/ml). The conversion of BSO to biotin
was performed as described for the reverse-phase HPLC analysis of the
BSO reductase reaction and the reaction products separated from the
protein by ultrafiltration. Following dilution with water, aliquots (25 µl) of each reaction product were applied to filter disks placed on
top of the minimal glucose plates containing E. coli
Mu29. The plates were incubated at 37 °C overnight and
inspected for bacterial growth, detectable as the formation of red
colonies surrounding the filter circles.
BSO reductase activities were
routinely determined at 600 nm under anaerobic conditions at 25 °C
in 50 mM Tris buffer, pH 8, as the oxidation of either
MV (115 µM) or BV
(115 µM) in the presence of BSO (1.7 mM).
Concentrations of MV
or BV
were calculated using
extinction coefficients of 13 mM
1
cm
1 (
600 nm) (Thorneley, 1974
) and 13 mM
1 cm
1 (
555 nm)
(Lissolo et al., 1984
), respectively. Activities were expressed in units of micromole of BSO consumed per min/nmol of enzyme.
Kinetic parameters were derived using the software "Enzfitter" (Elsevier Biosoft, Ferguson, MO).
NADPH:BSOR and NADPH:FR activities, expressed as micromole of NADPH
consumed per min/nmol of enzyme, were determined at 340 nm in 50 mM Tris buffer, pH 8.0, in the presence of either NADPH (250 µM) and BSO (1.7 mM) or NADPH (250 µM) and Fe(CN)63 (630 µM), respectively. NADPH:BSOR activity was also
determined in the presence and absence of added E. coli or
R. sphaeroides cell lysate supernatant, prior to and
following dialysis of the supernatant. In addition, cell lysates were
also assayed for endogenous NADPH oxidation, both in the presence and
absence of BSO.
BSO reduction to biotin was also monitored at 533 nm using the
colorimetric reagent, p-dimethylaminocinnamaldehyde
(McCormick and Roth, 1970). GST activities were determined at 340 nm
and 25 °C in 25 mM MOPS buffer, pH 7, in the presence of
1-chloro-2,4-dinitrobenzene (1 mM) and GSSH (0.5 mM) (Habig et al., 1974
).
Expression of the GST-BSO Reductase Fusion Protein
The results of expression studies using E. coli
harboring the GST-BSO reductase expression vector are shown in Fig.
1. Optimal expression of the soluble form of the GST-BSO
reductase fusion protein was obtained using low concentrations of
isopropylthio--galactoside (0.1 mM) coupled with
bacterial culture at room temperature for 5 h. Elevated
temperatures or longer growth periods resulted in decreased production
of the soluble fusion protein and a corresponding increase in the
formation of inclusion bodies. Under optimum expression conditions and
compared to control cells containing the GST expression vector without
the BSO reductase gene, the GST-BSO reductase fusion protein could be
detected as the presence of an additional protein band of molecular
mass approximately 106 kDa in both the pellet (Fig. 1, lane
P) and soluble (Fig. 1, lane S) protein fractions. Quantitation of protein expression levels suggested that the soluble GST-BSO reductase fusion protein represented 2-3% of the total soluble protein while the insoluble form represented 8-10% of the
pellet fraction. In comparison, control cells containing the vector
alone produced a major, soluble, protein with a molecular mass of 26 kDa, that exhibited GST activity (data not shown).
BSO Reductase Isolation
Under optimal conditions, the yield of purified BSO reductase protein corresponded to 1 mg from 8 liters of cells (8 g). The individual steps utilized to purify the BSO reductase to homogeneity together with the respective yields are indicated in Table I. The initial affinity purification of the soluble E. coli cell extract using glutathione-agarose resulted in the isolation of a complex protein mixture that exhibited both GST activity and MV:BSOR activity indicating the presence of the required fusion protein. Examination of the protein composition of this sample indicated the presence of four proteins, detected as individual bands corresponding to molecular masses of 106, 70, 60, and 26 kDa, respectively, following SDS-PAGE analysis (Fig. 1, lane A, u). The high molecular mass band, corresponding to a molecular mass of 106 kDa, that occasionally appeared as a doublet following SDS-PAGE, was subsequently identified as the GST-BSO reductase fusion protein by a combination of Factor Xa proteolysis and its retention of both GST and MV:BSOR activity. However, this band was usually partially proteolyzed even without the addition of Factor Xa, evident as the presence of the small 26-kDa GST fragment. Following proteolysis with Factor Xa (Fig. 1, lane A, c), the 106-kDa band was converted to two bands with apparent molecular masses of 80 and 26 kDa, respectively, the former retaining MV:BSOR activity and the latter retaining GST activity. Direct analysis of the amino terminus of the 80-kDa protein yielded the sequence shown in Table II, confirming the identity of this protein as BSO reductase while the 26-kDa protein corresponded to the cleaved GST expression tag.
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|
Analysis of the affinity-purified GST-BSO protein complex indicated that two additional protein components, corresponding to molecular masses of 70 and 60 kDa, respectively, consistently co-purified with the fusion protein. These two additional proteins were tightly associated since they were only partially resolved by fast protein liquid chromatography gel filtration of the complex (Fig. 1, lanes B and C).
To confirm that the two auxiliary proteins bound specifically to BSO
reductase and not the intact fusion protein, the isolated GST-BSO
reductase fusion protein complex (Fig. 2, lane
A) was treated with Factor Xa and affinity purified using
glutathione-agarose. The flow-through from the column contained BSO
reductase, identified by the retention of MV:BSOR activity, and both
the 70- and 60-kDa auxiliary proteins (Fig. 2, lane B),
whereas the cleaved GST portion remained bound to the column and could
subsequently be eluted with GSSH as the sole protein (Fig. 2,
lane C). BSO reductase could be resolved from the auxiliary
proteins by ion exchange chromatography yielding a homogeneous enzyme
(Fig. 2, lane D).
The purity of the isolated BSO reductase obtained following ion exchange and gel filtration chromatography was also examined using reverse-phase HPLC. The HPLC chromatogram indicated the presence of a single protein peak corresponding to an elution time of 40.5 min (data not shown).
Auxiliary Protein Identification
To identify the 60-kDa auxiliary protein that bound to BSO
reductase, the amino terminus of the protein was examined yielding the
sequence shown in Table II. This sequence was identical to the first 15 residues (AAKDVKFGNDARVKM) of the E. coli groEL protein (Hemmingsen et al., 1988). In contrast, we were unable to
obtain any reliable sequence from the amino-terminal region of the
70-kDa protein. However, following proteolysis, three internal peptides were obtained which yielded the sequences shown in Table II, confirming the identity of this protein as E. coli hsp70 (Bardwell and
Craig, 1984
) and corresponding to residues 93-106 (IIAADNGDAWVEVK),
587-594 (KMQELAQV) and 598-616 (LMEIAQQQHAQQQTAGADA),
respectively. Attempts to increase the level of soluble BSO reductase
expression, by co-transformation of the JM109 cells with the
groEL gene, failed to increase production of the soluble
fusion protein-chaperonin complex.
Spectroscopic Analysis
The UV-visible spectral characteristics of the native and SDS-treated BSO reductase and a sample of purified, oxidized R. sphaeroides Me2SO reductase are shown in Table III. Oxidized BSO reductase exhibited a protein peak at 278 nm with a broad shoulder centered around 370 nm with very little absorbance at higher wavelengths indicating the absence of any additional chromophores. Denaturation of both enzymes resulted in an increased intensity for both the dominant protein peak at 278 nm and increased resolution of the Mo cofactor peak, detected at approximately 375 nm, when compared to the spectra obtained for the native enzymes, consistent with cofactor release.
|
UV CD spectra of both BSO reductase and Me2SO reductase
were very similar and typical of proteins of the /
class. BSO
reductase exhibited a positive CD maximum at 192 nm and negative CD
maxima at 209 and approximately 219 nm, respectively, whereas
Me2SO reductase exhibited CD maxima at 192, 209, and 221 nm, respectively.
Cofactor Analysis
To identify the Mo cofactor bound to the recombinant BSO
reductase, the purified enzyme (Fig. 2, lane D) was
denatured with SDS and the cofactor isolated. The UV-visible absorbance
spectrum of the isolated Mo cofactor is shown in Fig. 3,
together with the spectrum obtained from a corresponding sample of
R. sphaeroides Me2SO reductase, a known
MGD-containing enzyme (Johnson et al., 1990). The spectrum
of the isolated cofactor exhibited an additional peak at 249 nm due to
absorption by the 5
-GMP portion of the cofactor. Identical results
have previously been reported for Me2SO reductase by
Johnson et al. (1990)
.
To confirm the identity of the Mo cofactor as MGD, fluorescence
emission spectra of the isolated cofactor, both prior to and following
pyrophosphatase cleavage, were obtained and compared to those from a
sample of cofactor isolated from Me2SO reductase. Incubation of the liberated MGD cofactor with pyrophosphatase, resulting in hydrolysis of the pyrophosphate linkage between the molybdopterin and 5-GMP moieties, produced an increase in the fluorescence intensity (Table IV) of both samples with
no shift in the fluorescence emission maxima which were determined to
be 469 nm for the cofactor isolated from BSO reductase and 468 nm for
MGD isolated from Me2SO reductase.
|
Enzyme Activities
HPLC AnalysisThe ability of the isolated BSO reductase to
convert BSO to biotin, utilizing MV as an electron donor, was
initially examined using reverse-phase HPLC analysis of the reaction
products, as shown in Fig. 4. Under the conditions used
for analysis, standard samples of BSO and biotin eluted at 9.2 min
(peak 1, Trace C) and 18.7 min (peak 2, Trace B), respectively, while
the large early peak present in all the elution profiles was due to the presence of MV. In the absence of BSO reductase or in the presence of
heat-inactivated enzyme, none of the substrate was reduced to biotin
(Traces C and D, respectively). However, in the presence of BSO
reductase (Trace E), approximately 50% of the BSO was converted to
biotin as evident by the decrease in the amplitude of the peak at 9.2 min and the appearance of the peak at 18.7 min due to biotin formation.
HPLC analysis was also utilized to examine the conversion of BSO to biotin catalyzed by BSO reductase using reduced glutathione and reduced thioredoxin as potential electron donors. Incubation of BSO reductase (5 µg) and BSO (1.6 mM) for 2 h at 25 °C in 50 mM Tris buffer, pH 8, in the presence of reduced glutathione (10 mM) or reduced thioredoxin (1 mM) and EDTA (1 mM), respectively, followed by HPLC analysis of the reaction mixture failed to indicate the formation of biotin, suggesting both reducing agents failed to function as suitable electron donors for BSO reductase.
Disk Microbiological Assay
To confirm that the BSO reductase reaction product eluting at 18.7 min on reverse-phase HPLC was biotin, we utilized this reaction product
as well as control samples of BSO and biotin, in the disk biological
assay as shown in Fig. 5. E. coli Mu29 mutants were unable to grow utilizing either the products of the reaction between MV
and BSO (Disk c), the products of the
reaction between MV
, BSO, and heat-inactivated enzyme (Disk
a), MV
(Disk d) or BSO in water (Disk f). In contrast, the
Mu29 mutants grew, visible as red halos surrounding the disc, in
either the presence of the products of the reaction between
MV
, BSO, and BSO reductase (Disk b), MV
and biotin
(Disk e), or biotin in water (Disk g). Thus the test organism grew well
only around the two disks containing authentic biotin or the product of
the BSO reductase-catalyzed reaction.
Initial Rate Kinetics
The results of the previous two assays
indicated that MV could function as a facile electron donor
for BSO reductase and therefore could also be used in the first direct
spectrophotometric assay for BSOR activity. Analysis of the initial
rate measurements of the consumption of MV
in the presence of
BSO and several alternative electron acceptors yielded the kinetic
constants shown in Table V.
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Under the conditions of the assay, all Lineweaver-Burk plots were
linear over the entire range of donor and acceptor substrate concentrations examined except for assays performed in the presence of
high concentrations of BSO (>2 mM), nicotinamide
N-oxide (>5 mM), and methionine sulfoxide
(>2.5 mM) where pronounced substrate inhibition was
observed. At pH 8.0 and using 50 mM Tris buffer, BSO
reductase yielded a Vmax of 0.9 µmol of BSO
consumed/min/nmol enzyme and Km values of 29 and 15 µM for MV and BSO, respectively. MV
could be replaced by BV
with no change in
Vmax (0.9 µmol of BSO consumed/min/nmol of
enzyme) but an increase in Km to 35 µM, suggesting the enzyme exhibited very little
specificity with respect to the nature of the artificial electron
donor. However, the enzyme could also utilize additional oxidizing
substrates including nicotinamide N-oxide, methionine sulfoxide, trimethylamine N-oxide, and Me2SO
with comparable rates to that obtained with BSO although the
Km values for these substrates varied from a low of
20 µM (nicotinamide N-oxide) to a high of 14.4 mM (Me2SO). However, values for
Vmax/Km indicated BSO
reductase exhibited a marked preference for BSO as the oxidizing
substrate.
Equivalent kinetic constants were also obtained for the MV:BSOR activity of the isolated GST-BSO reductase fusion protein indicating that the GST domain had no influence on the catalytic activity of the BSO reductase domain.
NAD(P)H-dependent BSO Reductase Activity
The presence of a putative ATP/GTP-binding P-loop in the BSO
reductase sequence (Pollock and Barber, 1995), prompted the examination of reduced pyridine nucleotides as potential electron donors for the
enzyme. Under aerobic conditions and in the absence of BSO, consumption
of either NADPH or NADH by BSO reductase was not observed. In contrast,
in the presence of BSO, rapid consumption of NAD(P)H was detected
resulting in the conversion of BSO to biotin which was subsequently
confirmed by both HPLC analysis and the disk microbiological assay of
the reaction mixture. BSO reduction was dependent on the presence of
the enzyme since in its absence or in the presence of heat-inactivated
enzyme, no NADPH was consumed or biotin produced. The results of
initial rate studies at pH 8.0, shown in Table VI,
indicated that the purified enzyme exhibited a NADPH:BSOR specific
activity of 30 µmol of NADPH consumed/min/nmol of enzyme with
Km values of 269 and 524 µM for NADPH and BSO, respectively. BSO reductase was also capable of utilizing NADH
as an electron donor, although with greatly reduced efficiency, the
specific activity being reduced approximately 11-fold from that
determined with NADPH while the Km for NADH was increased to 394 µM. NADPH also functioned as a suitable
electron donor for the reduction of a variety of alternative oxidizing substrates (Table VI) although BSO remained the preferred substrate. Identical studies performed using purified R. sphaeroides
Me2SO reductase revealed that in contrast to BSO reductase,
Me2SO reductase did not exhibit any reduced pyridine
nucleotide dependent activity.
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To determine if auxiliary proteins present in E. coli or R. sphaeroides cell lysates could enhance the activity of the purified BSO reductase, activities were examined in the presence and absence of either E. coli or R. sphaeroides cell extracts. Undialyzed cell lysates were observed to inhibit NADPH:BSOR activity approximately 2-fold, however, dialysis of the lysates to remove low molecular weight species resulted in no inhibition nor stimulation of enzyme activity, suggesting that in contrast to the E. coli BSO reductase the R. sphaeroides enzyme did not require any auxiliary proteins for functionality.
In the presence of NADPH, BSO reductase was also able to utilize
ferricyanide as an artificial electron acceptor. At pH 8.0, the enzyme
exhibited a NADPH:FR specific activity of 1.7 µmol of NADPH
consumed/min/nmol of enzyme with Km values of 16 and
662 µM for NADPH and
Fe(CN)63, respectively.
The preceding results demonstrate the first successful heterologous expression and isolation to homogeneity of a functional BSO reductase that contains MGD as its sole prosthetic group and which is catalytically active in the absence of any auxiliary protein components. In addition, we have provided the first demonstration that the enzyme can directly utilize reduced pyridine nucleotides as a source of reducing equivalents and also obtained initial rate kinetic constants that indicate that BSO reductase can utilize a variety of alternate oxidizing substrates in addition to d-biotin d-sulfoxide.
Expression of a recombinant BSO reductase as a GST fusion protein
presented several potential advantages for the production of a
functional molybdoprotein. First, the system allowed the affinity
purification of the BSO reductase under reducing conditions which could
be important when cells are broken by sonication to prevent damage of
the Mo cofactor due to oxidation by free radicals, while second,
glutathione-agarose has previously been used to isolate an active form
of the Mo cofactor from xanthine oxidase under reducing conditions
(Mendel and Alikulov, 1983) and therefore may stabilize the functional
forms of Mo-containing enzymes during purification.
While the cloning of the BSO reductase has not resulted in very high levels of expressed soluble enzyme since the majority of the BSO reductase is produced as inclusion bodies, it has facilitated the isolation and purification of the enzyme to homogeneity in an active form in quantities sufficient for the first spectroscopic and kinetic characterization of the Rhodobacter enzyme.
The isolation of a homogeneous BSO reductase has facilitated both
cofactor analysis and limited structural comparisons. The enzyme has
been demonstrated to contain MGD, which has also been shown to be the
Mo cofactor present in R. sphaeroides Me2SO
reductase (Johnson et al., 1990) and a number of other
related E. coli Mo-containing enzymes (Rajagopalan and
Johnson, 1992
). In addition, the spectroscopic and reverse-phase HPLC
analyses of the purified enzyme have indicated the absence of any
additional chromophores, confirming MGD to be the sole prosthetic
group. Comparison of the UV CD spectra obtained for BSO reductase and
Me2SO reductase indicates the two proteins have similar
conformations, which is also reflected in the extensive sequence
conservation (Barber et al., 1995
). These results are
supported by the recently determined x-ray structure of R. sphaeroides Me2SO reductase which has confirmed that
this enzyme contains MGD as its sole prosthetic group and identified it
as a mixed
/
protein in terms of its secondary structure (Schindelin et al., 1996
).
Previous work on the E. coli BSO reductase resulted in the
isolation of enzyme of undetermined purity (del Campillo-Campbell et al., 1979) while measurements of enzyme activity were
limited to the use of the disk biological assay, performed in the
presence of added cell lysate due to requirements for several auxiliary proteins. In contrast, the recombinant R. sphaeroides BSO
reductase has been demonstrated to be fully functional in the absence
of any additional auxiliary proteins. Comparison of the specific activity reported for the E. coli BSO reductase with that of
the R. sphaeroides enzyme suggests that the latter is
significantly more active (the specific activity reported for the
E. coli enzyme corresponds to 1.6 × 10
5
µmol of BSO consumed/min/nmol of enzyme while the corresponding value
for the R. sphaeroides enzyme is 9 × 10
1
µmol of BSO consumed/min/nmol of enzyme). However, this substantial difference may be partly due to the greater purity of the latter enzyme
and the use of an improved assay. While the enzyme was unable to
utilize low molecular weight thiol compounds as electron donors, in
agreement with previous genetic studies of E. coli BSO
reductase (del Campillo-Campbell et al., 1979
), reduced
viologens were found to act as efficient reductants.
The isolation of a functional BSO reductase has been confirmed by three
different assays: the direct analysis of the reaction products using
reverse-phase HPLC, the disk microbiological assay, which indirectly
confirms the presence of biotin as indicated by the growth of the test
organism, and the development of a direct spectrophotometric assay for
the enzyme-catalyzed oxidation of MV in the presence of BSO,
similar to the method used to assay the activity of Me2SO
reductase (Bastian et al., 1991
). The recombinant expression
and isolation of a functional Mo-containing enzyme has previously only
been described for rat hepatic sulfite oxidase (Garrett and
Rajagopalan, 1994
). The enzyme was expressed at low levels in E. coli, contained MPT, and retained sulfite:cytochrome c
reductase activity, although no specific activities were reported. In
contrast, recent attempts to express R. sphaeroides
Me2SO reductase in E. coli resulted in the
production of the appropriately-sized insoluble recombinant protein
which was devoid of Me2SO reductase activity (Hilton and
Rajagopalan, 1996a
).
In contrast to either E. coli BSO reductase or R. sphaeroides Me2SO reductase, we have demonstrated that
R. sphaeroides BSO reductase can directly utilize reduced
pyridine nucleotides as electron donors. Analysis of initial rate data
has indicated a marked preference for NADPH when compared to NADH, the
former yielding values for
Vmax/Km directly comparable
to those obtained using MV as the source of reducing
equivalents. While both purified Me2SO reductase and
partially-purified E. coli BSO reductase are unable to
directly use either NADPH or NADH as suitable reducing agents, the
E. coli BSO reductase has been shown to exhibit limited
NADPH-dependent BSOR activity in the presence of an
auxiliary protein referred to as protein-S2 reductase (del
Campillo-Campbell et al., 1979).
This work has provided the first information concerning the oxidizing
substrate specificity of BSO reductase. While the enzyme functions most
efficiently with BSO, nicotinamide N-oxide, methionine sulfoxide, trimethylamine N-oxide, and Me2SO
could also be utilized as substrates although with varying
efficiencies. While the enzyme was found to utilize nicotinamide
N-oxide with a degree of efficiency comparable to that of
BSO, markedly reduced efficiencies were observed for the remaining
substrates. Although the turnover numbers for all five oxidizing
substrates were comparable, the smaller substrates did not appear to
bind as tightly to the enzyme as the larger or heterocyclic compounds.
In addition, a similar substrate specificity was observed when using
either MV or NADPH as the reducing agent. The
MV
-dependent substrate specificity of BSO reductase appears to be the reverse of that for R. sphaeroides Me2SO reductase where substrates such as
trimethylamine N-oxide have been shown to bind less tightly
than Me2SO (Satoh and Kurihara, 1987
). Comparison of the
specific activities previously reported for R. sphaeroides
Me2SO reductase (Bastian et al., 1991
; Hilton and Rajagopalan, 1996b
) with that obtained for BSO reductase indicates that the two enzymes have comparable turnover numbers when utilizing Me2SO or BSO as oxidizing substrates, respectively. In
addition, our kinetic studies have indicated that BSO reductase can
reduce methionine sulfoxide to methionine at a rate approximately
300-fold greater than that obtained for a similar reaction catalyzed by the E. coli enzyme, peptide methionine sulfoxide reductase
(Rahman et al., 1992
), that has been postulated to provide a
repair mechanism for proteins that have been inactivated by oxidation.
Thus, the ability of BSO reductase to reduce a variety of oxidized
substrates may provide an additional pathway to reverse the results of
oxidative damage.
Previous studies of protein expression in E. coli have
indicated the involvement of chaperonins in promoting protein folding and inhibiting degradation of foreign proteins by
ATP-dependent proteolysis. Thus the expression of a mutant
form of alkaline phosphatase (pho61) in E. coli has
demonstrated the association of the mutant protein with both hsp70 and
grpE chaperonins (Sherman and Goldberg, 1992). For the expression of
the fusion protein, CRAG, which contained sequences of cro, protein A,
and a 14-amino acid portion of the lacZ gene product, the
fusion protein was found to exist in two complexes with either hsp70
(Hellebust et al., 1989
) or groEL (Sherman and Goldberg,
1991
). groEL has also previously been shown to co-purify with a
recombinant form of a plant glucosidase produced as a GST fusion
protein (Keresztessy et al., 1996
). In contrast, expression
of BSO reductase has demonstrated the formation of a specific complex
with both hsp70 and groEL and that the GST-BSO reductase fusion
protein-chaperonin complex retained activity comparable to that of the
purified enzyme suggesting retention of the native conformation in the
fusion protein complex. While the precise role of these auxiliary
chaperonin proteins in the expression of BSO reductase has not been
identified, they may potentially function to assist in the correct
folding or assembly of the recombinant protein.
We are grateful to Dr. Peter Neame for assistance with peptide sequencing.