From the Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710
Received for publication, August 11, 2000, and in revised form, October 20, 2000
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ABSTRACT |
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We were able to reconstitute molybdopterin
(MPT)-free sulfite oxidase in vitro with the molybdenum
cofactor (Moco) synthesized de novo from precursor Z and
molybdate. MPT-free human sulfite oxidase apoprotein was obtained by
heterologous expression in an Escherichia coli mutant with
a defect in the early steps of MPT biosynthesis. In vitro
reconstitution of the purified apoprotein was achieved using an
incubation mixture containing purified precursor Z, purified MPT
synthase, and sodium molybdate. In vitro synthesized MPT
generated from precursor Z by MPT synthase remains bound to the
synthase. Surprisingly, MPT synthase was found capable of donating
bound MPT to MPT-free sulfite oxidase. MPT was not released from MPT
synthase when either bovine serum albumin or Moco-containing sulfite
oxidase was used in place of aposulfite oxidase. After the inclusion of
sodium molybdate in the reconstitution mixture, active sulfite oxidase
was obtained, revealing that in vitro MPT synthase and
aposulfite oxidase are sufficient for the insertion of MPT into sulfite
oxidase and the conversion of MPT into Moco in the presence of high
concentrations of molybdate. The conversion of MPT into Moco by
molybdate chelation apparently occurs concomitantly with the insertion
of MPT into sulfite oxidase.
Sulfite oxidase belongs to the family of molybdenum cofactor
(Moco)1-containing enzymes
characterized by the presence of a mononuclear molybdenum coordinated
to the unique dithiolene group of a pterin derivative named
molybdopterin (MPT). The overall reaction catalyzed by most
Moco-containing enzymes, including sulfite oxidase, involves the
transfer of an oxygen atom between the substrate and water in a
two-electron oxidation-reduction reaction. Sulfite oxidase is
ubiquitous among animals and catalyzes the physiologically vital
oxidation of sulfite to sulfate, the terminal reaction in the oxidative
degradation of the sulfur-containing amino acids cysteine and
methionine. The enzyme is a homodimer located in the mitochondrial
intermembrane space. Each 52-kDa subunit of the human enzyme contains a
small N-terminal heme domain and a large C-terminal Moco-binding domain
(1). Genetic deficiency of sulfite oxidase, which can be caused by
either a mutation in its structural gene (isolated sulfite oxidase
deficiency) or a defect in the synthesis of Moco (molybdenum cofactor
deficiency), results in neurological abnormalities and often leads to
death at an early age (2).
The basic structure of Moco has been shown to be identical in all
organisms, and its biosynthetic pathway seems to be conserved because
genes encoding highly homologous proteins for Moco biosynthesis have
been found in archaea, bacteria, higher plants, Drosophila, and higher animals including humans. Biosynthesis of Moco has been studied most extensively in Escherichia coli, and
several genetic loci (moa, mob, mod,
moe, and mog) have been implicated in the
pleiotrophy of the molybdenum enzymes, most of them being involved in
the biosynthesis of Moco (3). The reactions of the Moco biosynthetic
pathway comprise three stages that are identical in all organisms using
molybdoenzymes: (i) conversion of a guanine nucleotide into the
meta-stable precursor Z, (ii) conversion of precursor Z into MPT, and
(iii) insertion of molybdenum into MPT, thus forming Moco. However, in
most bacteria an additional stage in the biosynthetic pathway involves
further modification of Moco leading to the formation of dinucleotide
variants of MPT containing GMP, CMP, AMP, or IMP (4). Conversion of
precursor Z to MPT requires the opening of a cyclic phosphate to
produce a terminal monoester as well as the transfer of sulfur to
generate the dithiolene group essential for molybdenum ligation (5).
Pitterle et al. (6) demonstrated that incubation of purified
precursor Z with MPT synthase resulted in the formation of MPT. MPT
synthase forms a heterotetramer composed of two small MoaD subunits
(8.5 kDa each) and two large MoaE subunits (16.8 kDa each). In
the activated form of MPT synthase, the C terminus of the small subunit
is converted to a glycine thiocarboxylate that acts as the sulfur donor
for the conversion of precursor Z to MPT. In turn, MPT synthase is resulfurated by the MoeB protein designated as MPT synthase sulfurase. The involvement of proteins analogous to MoaD and MoeB in pathways leading to the biosynthesis of thiamine and ferredoxin led to the
proposal that those pathways also operate by the thiocarboxylate mechanism (3). In the case of the ThiS protein, mass spectrometric evidence for a C-terminal glycine thiocarboxylate has been presented (7). Based on the observation that high concentrations of molybdate in
the growth medium can partially rescue a mogA mutant, the
MogA protein has been suggested to catalyze the in vivo
insertion of molybdenum into MPT (8).
Free Moco is extremely unstable (9) and thus cannot be used in studies
on the reconstitution of Moco-free proteins. In this paper, we describe
an in vitro system for the incorporation of nascent Moco
into Moco-free human sulfite oxidase expressed and purified from an
E. coli strain with a mutation in the early steps of Moco
biosynthesis. We show that MPT, produced in vitro from
precursor Z by MPT synthase and stabilized by tight binding to the
synthase, can be incorporated into aposulfite oxidase in the absence of
molybdate and without the requirement of any other proteins. We were
also able to reconstitute sulfite oxidase activity by the inclusion of
molybdate in the in vitro system. These results reveal that
no other proteins are required for the in vitro insertion of
MPT into sulfite oxidase or for the conversion of MPT into Moco under
the conditions used in the reconstitution mixture.
Bacterial Strains--
E. coli chlAI, chlN, and
chlM strains, now designated moaA Gel Electrophoresis--
Analytical polyacrylamide gel
electrophoresis was carried out in a discontinuous gel system (11)
using 4-20% nondenaturing gradient polyacrylamide gels (Bio-Rad). Gel
filtration molecular mass standards were obtained from Bio-Rad.
Purification of the Reaction Components--
Precursor Z was
isolated from E. coli moaD Enzyme Assays--
Sulfite oxidase activity was assayed at room
temperature by monitoring the reduction of cytochrome c at
550 nm (15) or ferricyanide at 420 nm (16) using a Shimadzu 1601 spectrophotometer. One unit of sulfite oxidase activity is defined as
an absorbance change ( In Vitro Reconstitution of Sulfite Oxidase Activity--
MPT was
produced in vitro by the method described by Pitterle
et al. (6) using purified precursor Z in 10 mM
sodium citrate buffer, pH 3, that was adjusted to pH 7.2 with NaOH, and
MPT synthase in 100 mM Tris, pH 7.2. For a standard
reconstitution assay, 30 nmol of precursor Z and 100 µg of MPT
synthase were allowed to react at room temperature in the presence or
absence of 20 mM sodium molybdate. After 15 min, 12 µg of
sulfite oxidase in 100 mM Tris, pH 7.2, was added, and the
reaction mixture, in a total volume of 80 µl, was incubated at room
temperature for 30 min before aliquots were taken and either analyzed
for MPT content or assayed in appropriate dilutions for sulfite oxidase activity.
MPT Analysis by Generation of Form A (Dephospho)--
To analyze
the amount of MPT present in sulfite oxidase or MPT synthase, the
purified protein was subjected to a procedure that converts MPT to the
stable oxidized fluorescent degradation product form A by treatment of
the protein at pH 2.5 in the presence of iodine at room temperature, as
originally described by Johnson et al. (17). After treatment
with alkaline phosphatase, dephospho form A was identified by HPLC
analysis at room temperature at a flow rate of 1 ml/min with an Alltech
C18 HPLC column equilibrated in 50 mM ammonium acetate,
10% methanol. Form A was assayed by monitoring its fluorescence with
an excitation at 297 nm and emission at 440 nm. All HPLC analyses were
performed using the Hewlett Packard 1090 solvent delivery system, and
eluting material was monitored either for absorbance using a Hewlett
Packard 1040A diode array detector or for fluorescence using a Hewlett
Packard 1046 fluorescence detector.
Purification of Recombinant Human Sulfite Oxidase from an E. coli
moaA In Vitro Synthesis of MPT from Precursor Z Using Active MPT
Synthase--
To synthesize MPT in vitro, purified
precursor Z and purified MPT synthase were incubated at room
temperature as described by Pitterle et al. (6). Purified
precursor Z was obtained from cells that contain a mutation in
moaD and thus accumulate the precursor (12). Active MPT
synthase was purified after expression in BL21 cells (13). Inactive
recombinant MPT synthase was purified from a
moeB In Vitro Reconstitution of Human Sulfite Oxidase--
E.
coli MPT synthase was previously shown capable of donating bound
MPT to the inactive aponitrate reductase present in Neurospora crassa nit-1 extracts (6). To test whether MPT synthase is also
capable of donating MPT to human sulfite oxidase, purified aposulfite
oxidase was incubated with active MPT synthase and precursor Z under
aerobic conditions. MPT synthase and precursor Z were incubated for 15 min at room temperature prior to the addition of aposulfite oxidase.
Sulfite oxidase activity was analyzed after a 30-min incubation of the
enzyme with the mixture of MPT synthase/precursor Z. Somewhat
unexpectedly, the inclusion of sodium molybdate in the in
vitro incubation mixture prior to the addition of sulfite oxidase
gave rise to sulfite oxidase activity (Table
I). The specific activity of
reconstituted sulfite oxidase was about 50% that of native sulfite
oxidase (Table I). In contrast, incubation of aposulfite oxidase with
MPT synthase and precursor Z without the addition of molybdate or with
inactive MPT synthase and precursor Z did not reconstitute sulfite
oxidase activity.
To determine whether the maximum of 50% reconstitution of sulfite
oxidase activity was due to limited incorporation of MPT into the
enzyme or limited molybdate insertion into MPT, an analysis of the
amount of cofactor present in reconstituted sulfite oxidase was carried
out. Purified His-tagged aposulfite oxidase was added to the
reconstitution mixtures in the presence or absence of sodium molybdate.
After incubation as described above, sulfite oxidase was isolated by
Ni-NTA chromatography. To determine the amount of MPT present in
reconstituted active and inactive sulfite oxidase as well as native
sulfite oxidase, the proteins were subjected to iodine oxidation to
produce the form A derivative of the cofactor. HPLC analysis revealed
that the amount of form A obtained from reconstituted active sulfite
oxidase (Fig. 3D) was about
57% of that obtained from native sulfite oxidase (Fig. 3A).
Surprisingly, the same amount of form A was obtained from inactive
sulfite oxidase after incubation with MPT synthase and precursor Z in
the absence of molybdate (Fig. 3C). These results indicate
that MPT can be incorporated into sulfite oxidase even in the absence
of molybdate. As expected, no form A could be obtained from aposulfite
oxidase treated with inactive MPT synthase and precursor Z (Fig.
3B). Because the enzyme activity obtained from reconstituted
sulfite oxidase correlated with the amount of cofactor present in the enzyme, it can be concluded that the less than maximal reconstitution of activity is due to the correspondingly lesser incorporation of MPT
and that the molybdenum content of the reconstituted enzyme corresponds
to the MPT content.
Time-dependent Reconstitution of Sulfite Oxidase
Activity--
To determine the effect of incubation time on the
reconstitution of sulfite oxidase with Moco, a time course experiment
was carried out. Sulfite oxidase was added under aerobic conditions to
a mixture of MPT synthase, precursor Z, and sodium molybdate, which
were preincubated for 15 min at room temperature. Aliquots of the
aerobic incubation mixture were assayed at specific intervals for
sulfite oxidase activity. Following the addition of sulfite oxidase
(Fig. 4), maximum sulfite oxidase
activity was achieved 10 min after the addition of sulfite oxidase to
the in vitro system. Longer incubation times failed to yield
a higher than 50% reconstitution of enzyme activity compared with
native sulfite oxidase (Table I). Because Moco is known to be very
sensitive to aerobic oxidation, the effect of anaerobiosis on the
in vitro reconstitution assays was examined. Surprisingly,
generation of MPT and insertion of Moco into sulfite oxidase under
anaerobic conditions did not increase the extent of sulfite oxidase
reconstitution (data not shown). Under anaerobic as well as aerobic
conditions, maximum activation of sulfite oxidase was achieved after an
incubation time of 10 min and corresponded to reconstitution of about
50-60% of the enzyme molecules.
Effect of the Concentration of Precursor Z, MPT Synthase,
Molybdate, and Sulfite Oxidase on the in Vitro Reconstitution of
Sulfite Oxidase with Moco--
To determine whether any components of
the in vitro activation of sulfite oxidase were limiting for
obtaining complete reconstitution of sulfite oxidase, the dependence of
activation on the concentrations of the reactants was investigated. In
general, the reactivation mixtures consisted of 100 µg of MPT
synthase, 30 nmol of precursor Z, 12 µg of aposulfite oxidase, and 20 mM sodium molybdate. When the sodium molybdate
concentration was varied over a range of 0-50 mM, maximal
sulfite oxidase activity was attained at a concentration of 5 mM (Fig. 5A). When
precursor Z concentration was varied from 0 to 50 nmol, a maximum of
sulfite oxidase activity was obtained at a concentration of 6-8 nmol
(Fig. 5B). When MPT synthase concentration was varied over a
range of 0-250 µg, maximum reconstitution of sulfite oxidase was
achieved at a concentration of ~50 µg of the enzyme (Fig.
5C). Collectively, for the reconstitution of 12 µg of
sulfite oxidase present in the in vitro activation system, none of the components were limiting under the standard conditions. As
shown in Fig. 5D, sulfite oxidase concentrations greater
than 50 µg/reactivation mixture were required to produce a decrease in specific activity. Because this is approximately 4 times the amount
of sulfite oxidase used in the standard reconstitution assay, it can be
concluded that all components required for the in vitro
synthesis of Moco are present in optimum amounts in the in
vitro assay and that the extent of reconstitution cannot be increased with higher concentrations of any of the components of the
reconstitution mixture.
Analysis of the Mechanism of MPT Incorporation into Sulfite
Oxidase--
With the knowledge that MPT can be inserted into sulfite
oxidase in vitro, it was of further interest to study the
mechanism of insertion of MPT and molybdate into aposulfite oxidase. To determine whether molybdenum is incorporated into MPT at a nonprotein bound stage before the insertion of MPT into sulfite oxidase, the
stability of MPT produced by MPT synthase over time after the addition
of sodium molybdate was investigated. MPT synthase, precursor Z, and
sodium molybdate were preincubated under aerobic conditions for 15-75
min before the addition of sulfite oxidase. After sulfite oxidase was
added, the reactivation mixtures were incubated for 30 min at room
temperature before aliquots were removed and assayed for sulfite
oxidase activity. As shown in Fig. 6, the
ability to reconstitute sulfite oxidase activity did not decrease with
an increase in elapsed time before the addition of aposulfite oxidase.
Because MPT is very sensitive to air oxidation, these results reveal
that MPT remains bound to MPT synthase and is therefore protected from
air oxidation prior to its insertion into sulfite oxidase. To analyze
whether release of MPT from MPT synthase is specifically induced by
aposulfite oxidase or by a nonspecific protein interaction, bovine
serum albumin (BSA) was added to the preincubation mixture. As shown in
Fig. 6, the addition of BSA had little, if any, effect on sulfite
oxidase reconstitution. Therefore, it is very likely that MPT is
released from MPT synthase by a specific protein interaction with
sulfite oxidase.
To determine whether MPT is released from MPT synthase by interacting
nonspecifically with any form of sulfite oxidase or is only released
from MPT synthase for incorporation into the molybdopterin-free form of
sulfite oxidase, the MPT content of MPT synthase after incubation with
MPT-containing and MPT-free sulfite oxidase was analyzed. MPT synthase
was incubated with precursor Z and sodium molybdate for 15 min before
the addition of either His-tagged active sulfite oxidase purified from
a wild-type strain (14) or His-tagged MPT-free aposulfite oxidase
purified from the moaA Time-dependent Molybdenum and MPT Incorporation into
Sulfite Oxidase--
The data presented above demonstrate that MPT
exists only in a protein-bound stage and is transferred specifically to
MPT-free sulfite oxidase. To determine whether molybdenum is inserted
into MPT bound to MPT synthase before being transferred to
sulfite oxidase, precursor Z, MPT synthase, and sodium molybdate were incubated for 15 min at room temperature, and MPT synthase was purified
afterward over a gel filtration column to remove free molybdate.
Sulfite oxidase was added to the gel-filtered MPT synthase, and the
ability of MPT synthase to reconstitute sulfite oxidase activity
without the addition of extra molybdate was analyzed. No sulfite
oxidase activity was reconstituted under these conditions. However,
inclusion of molybdate in the reconstitution mixture did produce the
expected level of activity (data not shown). From these data it can be
concluded that MPT synthase is unable to bind molybdate or convert MPT
to Moco.
The ability to reconstitute demolybdo-sulfite oxidase with molybdate
in vitro was already observed by Jones et al.
(15), who reported that molybdate could reconstitute a maximum of 30% of the demolybdo-sulfite oxidase isolated from tungsten-treated rats.
To examine the conditions for molybdenum insertion into sulfite
oxidase-bound MPT, MPT synthase was preincubated with precursor Z for
15 min at room temperature, and molybdate was added either before or at
different time points after the addition of sulfite oxidase. After
molybdate was added, the reactivation mixtures were incubated for 30 min at room temperature. Aliquots were then removed and assayed for
sulfite oxidase activity. As shown in Fig.
8, the ability to reconstitute sulfite
oxidase activity decreased steadily with increasing time before
molybdate addition to the reconstitution mixture. The extent of
reconstitution of sulfite oxidase activity reached its minimum at 10 min and did not decrease further. These data show that optimal
reconstitution of sulfite oxidase is achieved when molybdate is added
to the reconstitution mixture either at the same time or prior to the addition of sulfite oxidase. It may also be concluded that molybdate is
inserted into MPT as soon as the latter is bound to sulfite oxidase,
but perhaps because a conformational change of sulfite oxidase after
the insertion of MPT, a less than maximal reconstitution with molybdate
is achieved when its addition is delayed from time of MPT transfer.
After showing that the molybdenum atom is inserted into MPT concomitant
with or prior to MPT insertion into sulfite oxidase, it was of further
interest to analyze whether time courses of cofactor handoff from MPT
synthase to sulfite oxidase differ in the presence and absence of
molybdate in the reconstitution mixture. For this purpose, MPT synthase
and precursor Z were incubated for 15 min at room temperature in
parallel sets with and without the addition of sodium molybdate. After
the addition of sulfite oxidase to the reconstitution mixture, the
transfer of MPT from MPT synthase to sulfite oxidase was inhibited at
different time points by the addition of 200 mM
iodoacetamide to convert MPT to its alkylated product,
carboxamidomethyl (cam) MPT. Iodoacetamide has been shown to act
on MPT bound to MPT synthase in the absence of denaturing agents (5).
In contrast, the cofactor bound to sulfite oxidase, which is buried
deeply in the enzyme, remains unaffected by the addition of
iodoacetamide. Because carboxamidomethyl (cam) MPT, in contrast
to form A, is nonfluorescent, it is possible to convert specifically
the MPT bound to sulfite oxidase to form A and thus identify the amount
of MPT present in sulfite oxidase without separating it from MPT
synthase. As shown in Fig. 9, HPLC analysis revealed that, within experimental error, the time course of
MPT transfer to sulfite oxidase was not significantly influenced by the
presence of sodium molybdate in the reconstitution mixture. In the
presence or absence of sodium molybdate (Fig. 9, A and B), a maximum incorporation of MPT into sulfite oxidase was
achieved at ~10 min after the addition of sulfite oxidase to the
in vitro system. These findings are in agreement with the
data obtained for the time-dependent reconstitution of
sulfite oxidase activity shown in Fig. 4. It can therefore be concluded
that molybdate does not affect the kinetics of MPT handoff from MPT
synthase to MPT-free sulfite oxidase.
In most reconstitution studies on cofactor-containing proteins,
the standard procedure is to obtain the apoprotein by a resolution procedure to remove the cofactor and use a source of the cofactor to
reconstitute the apoprotein. In the case of sulfite oxidase, this
approach is not possible because of the size of the protein, the
presence of two prostetic groups, and the extreme instability of
released Moco.
To obtain Moco-free sulfite oxidase, the cloned gene of human sulfite
oxidase was expressed in the Moco deficient
moaA As for a source of the extremely unstable cofactor, the data presented
here demonstrate that Moco can be synthesized in vitro by
ligation of molybdate to MPT generated de novo from
precursor Z and MPT synthase. This was a somewhat surprising
observation because genetic and biochemical studies in E. coli have implied the involvement of the MogA and MoeA gene
products in cofactor biosynthesis. It has been proposed that
molybdenum, which enters the cell as the stable oxyanion molybdate by a
high affinity molybdate transport system (19), undergoes some type of
modification prior to incorporation into MPT within the cell. For
example, a role for the MoeA protein in generating a thiomolybdenum
species that might be used in Moco biosynthesis has been suggested
(20). Our results show that in vitro, molybdate can be
inserted into MPT and reconstitute sulfite oxidase activity without
further modification by any other proteins. So far, E. coli
MogA has been proposed to act as a molybdochelatase incorporating
molybdenum into MPT in vivo (21), an observation based on
the finding that high concentrations of molybdate can partially rescue
a mogA mutant. Although MogA binds MPT tightly and a
putative binding pocket for MPT has been identified in the crystal
structure of E. coli MogA, binding of molybdate could not be
demonstrated for MogA to date (22, 23). Because the MogA protein is not
required for in vitro insertion of molybdate into MPT, its
function in the cell still remains unclear. It can only be speculated
that MogA might act as an MPT carrier or storage protein because MogA has a high affinity for MPT or that MogA might be required for the
insertion of molybdate into MPT under low molybdate concentrations, a
function not required in vitro with concentrations of sodium molybdate of 5 mM and higher.
The data presented above indicate that the in vitro
reconstitution of aposulfite oxidase proceeds as shown in Fig.
10. As already reported by Pitterle
et al. (6), MPT synthase is required for the conversion of
precursor Z to MPT, a reaction that can be carried out in
vitro with the purified components. MPT produced in
vitro by active MPT synthase remains bound to the system, whereas
inactive MPT synthase, lacking the thiocarboxylate at the terminal
glycine of the small subunit, is unable to convert precursor Z to MPT but does bind precursor Z tightly. Because MPT synthase was shown to be
incapable of binding molybdate or forming Moco, MPT must be released
from MPT synthase for the conversion of MPT into Moco. Free MPT has
previously been shown to be highly sensitive to aerobic oxidation.
Because all reactions for the successful in vitro
reconstitution of aposulfite oxidase were performed under aerobic
conditions, it would appear that MPT is transferred directly to
MPT-free sulfite oxidase so that MPT stays protein bound and is
therefore protected from aerobic oxidation. In our system, 50%
reconstitution of sulfite oxidase was achieved when molybdate was added
to the in vitro reconstitution mixture prior to or at the
same time as the addition of sulfite oxidase. After the insertion of
MPT into sulfite oxidase, the ability to reconstitute sulfite oxidase
activity with added molybdate decreased with time. This might indicate
that after MPT is incorporated into sulfite oxidase, the structure of
the enzyme changes such that conditions are no longer optimal for insertion of molybdate into sulfite oxidase. Because molybdate is very
similar to sulfate, it is possible that molybdate can be bound to the
substrate-binding site present in sulfite oxidase, (18) which enables
the insertion of molybdate into MPT even after a structural change of
the enzyme. Even though we could show that the cofactor handoff from
MPT synthase to sulfite oxidase is a specific reaction in which the
cofactor is released from MPT synthase only after incubation with
MPT-free sulfite oxidase, it still remains unclear whether this
reaction will occur in the same manner in vivo. In the
cell, it is likely that molybdate is inserted into MPT before its
incorporation into sulfite oxidase, a reaction that might be carried
out by the MogA protein. We could show that MPT synthase is also able
to pass MPT to the MogA protein (data not shown) so that in
vivo a possible pathway for cofactor handoff might be that MPT
synthase interacts with MogA, the molybdenum atom is inserted into
MogA-bound MPT, and the molybdenum-containing cofactor is inserted into
sulfite oxidase by the MogA protein.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,
moeB
, and moaD
, are
isogenic mutants that were described previously (10). E. coli BL21(DE3) cells were obtained from Novagen. The
DE3 lysogenization kit from Novagen was used to integrate the gene for T7
RNA polymerase into the chromosome of the E. coli
moeB
strain.
cells using
high-performance liquid chromatography (HPLC) with reverse phase and anion exchange columns (12). Cloned MPT synthase was expressed in a
pET15b vector (Novagen) in E. coli BL21(DE3) and
moeB
(DE3) cells, and the protein was purified
by ammonium sulfate precipitation and gel filtration according to the
procedure described by Rudolph et al. (13). The human
sulfite oxidase gene was cloned into a pTrc-His vector resulting in an
N-terminal fusion to a 6xHis-tag, expressed in E. coli
moaA
cells and purified by Ni-NTA
chromatography (14). For comparison, non-His-tagged human sulfite
oxidase was used, which was expressed and purified from E. coli as described by Temple et al. (14).
A) of 1/min.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Mutant--
To obtain human sulfite oxidase without
bound Moco, the enzyme was expressed heterologously in an E. coli
chlAI mutant strain. This strain contains a point mutation in the
moaA gene, which is required for the first step of Moco
biosynthesis, namely the conversion of a guanine nucleotide into
precursor Z. The enzyme was expressed as an N-terminal fusion to a
His-tag and purified by Ni-NTA chromatography as described by Temple
et al. (14). The absorption spectrum of the purified
inactive protein showed the typical spectrum of a cytochrome
b5, representing the N-terminal part of the
protein (data not shown). Native polyacrylamide gel electrophoresis
revealed that the inactive enzyme, without the molybdenum cofactor
bound to its C-terminal part, retained its ability to form a homodimer
with a molecular mass of ~110 kDa (Fig.
1).
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Fig. 1.
Analysis of native sulfite oxidase and
aposulfite oxidase by native polyacrylamide gel electrophoresis.
Purifed sulfite oxidase was electrophoresed under nondenaturing
conditions in 4-20% polyacrylamide gradient gels and stained for
protein with Coomassie Brilliant Blue. Lane 1 contains 3 µg of native sulfite oxidase, and lane 2 contains 3 µg
of His-tagged aposulfite oxidase purified from a
moaA mutant strain.
strain deficient in MPT synthase
sulfurylase activity (5). In agreement with the results obtained by
Pitterle et al. (6), MPT formed by incubation of active MPT
synthase with precursor Z remains bound to MPT synthase (Fig.
2). For these experiments, active or
inactive MPT synthase was incubated with precursor Z at room
temperature and dialyzed afterward to remove excess precursor Z. Subsequent heat treatment in the presence of acidic iodine released
form A, the oxidized fluorescent degradation product of MPT. HPLC
analysis revealed that form A could be obtained from active MPT
synthase (Fig. 2A), whereas no form A was obtained from
inactive MPT synthase (Fig. 2B). The fluorescence peak
eluting at 3.8 min from the C18 reverse phase HPLC column after iodine treatment of inactive MPT synthase was identified as compound Z, the
oxidized product of precursor Z (data not shown). Therefore, MPT
remains tightly bound to active MPT synthase after dialysis, whereas
inactive MPT synthase binds precursor Z tightly but is unable to
convert it to MPT because of the absence of the glycine thiocarboxylate
at the C-terminus of the small MoaD subunit.
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Fig. 2.
Analysis of fluorescent derivatives obtained
from active and inactive MPT synthase after incubation with precursor
Z. HPLC elution profiles of form A isolated from 300 µg of
active MPT synthase incubated with precursor Z (A) or 300 µg of inactive MPT synthase incubated with precursor Z
(B). Incubation mixtures contained 1.5 mg of active/inactive
MPT synthase and 300 nmol of precursor Z. After a 30-min incubation,
MPT synthase was desalted over a PD10 column equilibrated in 100 mM Tris, pH 7.2, and bound MPT was then converted into form
A as described under "Experimental Procedures."
Activity of native sulfite oxidase or in vitro reconstituted aposulfite
oxidase using cytochrome c or ferricyanide as electron acceptors
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Fig. 3.
Analysis of fluorescent derivatives of the
MPT cofactor from reconstituted sulfite oxidase. HPLC elution
profiles of form A isolated from 82 µg of native sulfite oxidase
(A), 82 µg of aposulfite oxidase incubated with inactive
MPT synthase and precursor Z (B), 82 µg of aposulfite
oxidase incubated with active MPT synthase and precursor Z
(C), and 82 µg of aposulfite oxidase incubated with active
MPT synthase, precursor Z, and 30 mM sodium molybdate
(D). Incubation mixtures originally contained 360 µg of
sulfite oxidase, 1.5 mg of MPT synthase, and 300 nmol of precursor Z. All incubations were carried out as described under "Experimental
Procedures" at room temperature in a total volume of 1 ml in 100 mM Tris, pH 7.2. His-tagged sulfite oxidase was purified
from the reactivation mixtures by Ni-NTA chromatography and dialyzed
against 100 mM Tris, pH 7.2. MPT or Moco was converted into
form A as described previously.
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Fig. 4.
Time dependent activation of sulfite oxidase
in vitro. 100 µg of MPT synthase, 30 nmol of
precursor Z, and 20 mM sodium molybdate were preincubated
for 15 min at room temperature before the addition of 12 µg of
sulfite oxidase. Sulfite oxidase activity was determined before
(time 0) or at different time points after the addition of
sulfite oxidase.
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Fig. 5.
Effects of reactant concentration on in
vitro reconstitution of aposulfite oxidase. For the
in vitro activation of sulfite oxidase, MPT synthase,
precursor Z, and sodium molybdate were preincubated for 15 min prior to
the addition of aposulfite oxidase. After the addition of sulfite
oxidase, the reactivation mixtures were incubated for another 30 min.
All incubations were carried out at room temperature in a total volume
of 80 µl in 100 mM Tris, pH 7.2. Dependence on sodium
molybdate concentration (A), dependence on precursor
concentration (B), dependence on MPT synthase concentration
(C), and dependence on sulfite oxidase
concentration(D). Standard reactivation mixtures contained
100 µg of MPT synthase, 30 nmol of precursor Z, 20 mM
sodium molybdate, and 12 µg of sulfite oxidase.
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Fig. 6.
Analysis of the stability of MPT produced by
MPT synthase and the influence of BSA on the in
vitro reconstitution of sulfite oxidase. 100 µg of
MPT synthase, 30 nmol of precursor Z, and 20 mM sodium
molybdate were preincubated for 15-75 min at room temperature before
the addition of 12 µg of sulfite oxidase (straight line
with circles). In a second set of reconstitution mixtures,
the influence of 10 µg of BSA on the in vitro
reconstitution was analyzed (dashed line with
triangles). BSA was added to the reconstitution mixtures
before sulfite oxidase. After the addition of sulfite oxidase, the
activation mixtures in 100 mM Tris, pH 7.2, were incubated
for 30 min at room temperature before sulfite oxidase activity was
determined.
mutant strain. After
the addition of sulfite oxidase, the reactivation mixtures were
incubated for 30 min at room temperature. To analyze the MPT content of
MPT synthase, the incubation mixtures were dialyzed and applied on a
Ni-NTA column to remove His-tagged sulfite oxidase. The flow-through
from the Ni-NTA column, containing MPT synthase, was collected,
dialyzed, and concentrated. Form A was generated from equal amounts of
MPT synthase by the method described under "Experimental
Procedures." As shown in Fig. 7, HPLC
analysis revealed that form A was obtained from MPT synthase incubated with cofactor-containing sulfite oxidase but not from MPT synthase, which was incubated with MPT-free sulfite oxidase. Therefore, MPT seems
to be released from MPT synthase in a specific interaction with the
MPT-free form of sulfite oxidase only.
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Fig. 7.
Analysis of form A obtained from MPT synthase
purified after incubation with MPT-containing or MPT-free sulfite
oxidase. HPLC elution profiles of form A isolated from 112 µg of
MPT synthase after incubation with His-tagged MPT-containing sulfite
oxidase (A) and 112 µg of MPT synthase after incubation
with His-tagged MPT-free sulfite oxidase (B). Incubation
mixtures contained 300 µg of MPT synthase, 150 nmol of precursor Z, 2 mM sodium molybdate, and 1 mg of sulfite oxidase. After a
30-min incubation, the reactivation mixture was dialyzed against 50 mM NaPi and 300 mM NaCl, pH 8.0, and applied to a Ni-NTA column to bind the His-tagged sulfite oxidase.
The flow-through from the column was collected and dialyzed against 100 mM Tris, pH 7.2. MPT synthase was concentrated to 280 µg/ml and then treated with acidic iodine to convert MPT into form
A.
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Fig. 8.
Time course for the molybdenum activation of
sulfite oxidase. 100 µg of MPT synthase and 30 nmol of precursor
Z were preincubated for 15 min at room temperature before the addition
of 12 µg of sulfite oxidase. Sodium molybdate (20 mM) was
added either concurrently with (0 min) or at different time points
after sulfite oxidase addition. Sulfite oxidase activity was determined
30 min after molybdate addition.
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Fig. 9.
Influence of molybdate on the
time-dependent MPT incorporation into sulfite oxidase.
360 µg of MPT synthase and 100 nmol of precursor Z were preincubated
for 15 min at room temperature without (A) or with the
addition of 50 mM sodium molybdate (B). After
the addition of 160 µg of sulfite oxidase, the reconstitution was
stopped at specified intervals by the addition of 200 mM
iodoacetamide. After a further 30-min interval, MPT or Moco was
converted into form A as described under "Experimental
Procedures." Each point demonstrates the relative fluorescence
of form A obtained from the peak area after HPLC analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
mutant of E. coli as a
His-tagged protein. The purified apoprotein contained stoichiometric
amounts of heme, showing that the absence of Moco does not prevent heme
binding to the N-terminal domain of sulfite oxidase. The data presented
in Fig. 1 also showed that the apoprotein is a dimer just like native
sulfite oxidase. The x-ray structure of the highly homologous chicken
sulfite oxidase revealed that the sulfite oxidase monomer actually
folds into three domains, the N-terminal heme domain, the central
domain containing Moco, and the C-terminal domain entirely responsible for the dimeric structure of sulfite oxidase (18). The fact that the
apoprotein of human sulfite oxidase is a dimer demonstrates that,
despite the absence of Moco in the central domain, the C-terminal domain folds in the correct manner to generate the dimer interface. The
data presented in this paper have shown that the central domain of the
apoprotein is sufficiently unfolded to be able to bind MPT and form
Moco in vitro. These findings support the conclusion that
each of the three domains of sulfite oxidase can attain its folded
structure independently of the other two domains.
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Fig. 10.
Model for the in vitro
reconstitution of sulfite oxidase with MPT and
molybdate.
Overall, the maximum activation of sulfite oxidase obtained in our in vitro system corresponded to reconstitution of about 50% of the enzyme molecules with Moco. It is likely that the isolated sulfite oxidase contains improperly folded molecules and only about 50% of enzyme molecules can be reconstituted. This finding is in agreement with the observation that aposulfite oxidase is extremely unstable, and maximum reconstitution of aposulfite oxidase was achieved only with freshly prepared enzyme.
In summary, the work presented above describes for the first time the in vitro reconstitution of aposulfite oxidase with MPT and molybdate. Sulfite oxidase deficiency in humans, which can be caused by either a mutation in the gene encoding sulfite oxidase alone or a mutation in one of the genes involved in Moco biosynthesis, results in neurological abnormalities and often leads to death at an early age. It is an autosomal recessive disease and no therapy is known to date. The in vitro reconstitution of sulfite oxidase, with Moco yielding in an active enzyme, might present a starting point for the reconstitution of sulfite oxidase activity in patients with mutations in the biosynthetic pathway of Moco.
A search of completed and incomplete genomes shows that the Moco
biosynthestic pathway has widespread phylogenetic existence and is
present even in pathogens such as Helicobacter pylori, Vibrio cholerae, Mycobacterium tuberculosis, and
several others. The roles of molybdoenzymes in these pathogens are
unknown, but one possibility is that the ability of molybdoenzymes to
hydroxylate or dehydroxylate certain compounds enables these pathogens
to detoxify such compounds. Indeed, recently Kozmin et al.
(24) have shown that the extreme sensibility of the E. coli
mutant (uvrB-bio) to 6-hydroxylaminopurine and other
base analogs is due to mutations in the Moco biosynthestic pathway.
This finding raises the possibility that inhibition of the Moco
biosynthetic pathway could provide an avenue for developing
bacteriocidal compounds to counter the effects of infection by bacteria
including pathogens. The reaction catalyzed by MPT synthase,
i.e. the conversion of precursor Z to MPT, is the best
defined step in the Moco biosynthetic pathway and, as demonstrated
here, can be accomplished in vitro. The present studies have
enabled the coupling of the reaction catalyzed by MPT synthase to the
activation of aposulfite oxidase, providing an extremely sensitive
assay for the activity of MPT synthase. In the future, this procedure
should prove valuable for assessing the potential pharmacological
usefulness of putative inhibitors of MPT synthase.
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ACKNOWLEDGEMENTS |
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We thank C. Temple and R. Wiley for the preparation of active sulfite oxidase and Dr. M. Wuebbens for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported financially by a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to S. L.) and additionally by Grant GM44283 from the National Institutes of Health (to K. V. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Tel.: 919-681-8845;
Fax: 919-684-5470; E-mail: raj@biochem.duke.edu.
Published, JBC Papers in Press, October 20, 2000, DOI 10.1074/jbc.M007304200
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ABBREVIATIONS |
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The abbreviations used are: Moco, molybdenum cofactor; MPT, molybdopterin; HPLC, high-performance liquid chromatography; BSA, bovine serum albumin; Ni-NTA, nickel-nitrilotriacetic acid.
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REFERENCES |
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